Treatment of tauopathies

ABSTRACT

The invention is directed to methods of treatment of Alzheimer&#39;s disease and other tauopathies, via the administration of antibodies having specificity to abnormal forms of tau protein, the antibodies showing no binding and/or reactivity to a normal tau protein and being administered under conditions and in amounts effective to prevent or treat Alzheimer&#39;s disease or other tauopathies. In certain embodiments, the antibodies are selective for soluble truncated tau protein truncated at (i) its C-terminus after the glutamic acid residue Glu391, or (ii) at the aspartic acid residue Asp421, or (iii) at its N-terminus at the aspartic acid residue Asp13, or (iv) a combination of (i)-(iii). Further aspects of the invention are directed to the administration of an immunogen comprising an abnormal tau, preferably a soluble truncated tau.

This application is a continuation of U.S. Ser. No. 14/989,627, filedJan. 6, 2016, which is a continuation of U.S. Ser. No. 13/363,292, filedJan. 31, 2012, which claims priority to U.S. Provisional ApplicationSer. No. 61/438,083, filed Jan. 31, 2011, the disclosures of which arehereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a common chronic progressiveneurodegenerative disease in which there is an irreversible loss ofcognitive and behavioral functions. The disease can persevere for over10 years, advancing from mild symptoms to extremely severemanifestations. AD is said to afflict approximately 10% of thepopulation over the age of 65 and more than 30% of the population overthe age of 80.

Pathologically, Alzheimer's disease presents as extracellular amyloidplaques and intracellular neurofibrillary tangles. The neurofibrillarytangles are composed, e.g., of the microtubule-binding protein tau,which is assembled into paired helical and straight filaments. It hasbeen suggested that these entities may be functionally linked, althoughthe mechanisms by which amyloid deposition promotes pathological taufilament assembly is not clear.

The common denominator of intracellular neurofibrillary structures(neurofibrillary tangles, dystrophic neurites, and neurophil threads) ispaired helical filaments (PHFs). The major protein subunit of the PHFsis microtubule associated protein tau in abnormally hyperphosphorylatedform (Grundke-Iqbal et al., 1986; Wischik et al., 1988 a,b). Neuronswith neurofibrillary changes degenerate, and the degree of thisdegeneration directly correlates with the degree of dementia in theaffected individuals (Blessed et al., 1968).

Normal tau is a microtubule associated protein that distributes mainlyto axons. Tau protein takes part in modulating the assembly, spatialorganization and behavior of microtubules (MT) in neurons and probablyglial cell bodies (Drewes et al., 1998; Drubin and Kirschner, 1986;Lo-Presti et al., 1995). Tau proteins are encoded by a single genelocated on chromosome 17, but are detected as multiple isoforms intissue extracts from adult brains (Goedert et al., 1989; Himmler A.,1989; Kosik et al., 1989). Heterogeneity of tau proteins is in part dueto alternative splicing, giving rise to six isoforms in the adult humanbrain. These distinct isoforms differ by the presence or absence of 29-or 58-amino acid inserts in the amino-terminal region and by theaddition or deletion of a tandem repeat (which can be repeated either 3or 4 times) in a carboxy-terminal region of tau referred to asmicrotubule binding domain. This region is composed of imperfect repeatsof 31 or 32 amino acid residues. Referring to the longest human tauprotein isoform, htau40, containing all inserts (441 amino acid long) inhumans, the smallest tau isoform contains 352 amino acid residues withthree tandem repeats in the MT-binding domain and no amino terminalinserts, whereas the largest isoform contains 441 residues with fourrepeats and both amino terminal inserts.

A number of neurological diseases are known to have filamentous cellularinclusions containing microtubule associated protein tau, e.g.,Alzheimer's disease (AD), progressive supranuclear palsy (PSP),corticobasal degeneration (CBD), Pick's disease (PiD) and a group ofrelated disorders collectively termed frontotemporal dementia withParkinsonism linked to chromosome 17 (FTDP-17), amyotropic lateralsclerosis (ALS), Creutzfeldt-Jakob disease (CJD), dementia pugilistica(DP), Gerstmann-Straussler-Scheinker disease (GSSD), Lewy body diseaseand Huntington disease (Dickinson et al., 1998; DiFiglia et al., 1997;Forno, 1986; Hirano and Zimmerman, 1962; Nishimura et al., 1995;Prusiner 1996; Reed et al., 1998; Roberts, 1998; Schmidt et al., 1996;Shankar et al., 1989; Spillantini et al., 1998). Although the etiology,clinical symptoms, pathologic findings and the biochemical compositionof inclusions in these diseases are different, there is emergingevidence suggesting that the mechanisms involved in aggregation ofnormal cellular proteins to form various filamentous inclusions arecomparable. It is believed, that an initial alteration in conformationof microtubule associated protein tau, that initiates generation ofnuclei or seeds for filament assembly, is one of the key features. Thisprocess can be influenced by the posttranslational modification ofnormal proteins, by mutation or deletion of certain genes and by factorsthat bind normal proteins and thus alter their conformation.

The tau protein is very hydrophilic, and is one of the most solubleproteins known. It can be readily extracted from brain tissue orcultured cells. Therefore, the aggregation of tau protein in AD ishighly suspicious. In comparison, filamentous tau extracted fromAlzheimer's diseased brain tissues is relatively insoluble. Besidesphosphorylation, insoluble and normal soluble tau differ in the extentof posttranslational modifications, which include glycosylation,glycation, ubiquitination and racemization (Kenessey et al., 1995; Ko etal., 1999; Mori et al., 1987; Wang et al., 1996; Yan et al., 1994).

It has previously been reported that tau in AD brain neurofibrillarydeposits is truncated at its C-terminus at the glutamic acid residueGlu391 (Novak, et al., 1989; Novak, et al., 1993). Truncation of tau atGlu391 leads to AD-specific conformational changes that are recognizedby the conformational antibody MN423 (Novak, et al., 1989; Novak, etal., 1993; Csokova, et al., 2006; Skrabana, et al., 2006; and Skrabana,et al., 2007).

The mechanism by which tau protein is modified to take part in filamentformation in AD is unknown. Phosphorylation of tau affects the potentialof tau to form aggregates, producing either stimulatory or inhibitoryeffects, presumably depending on the site of phosphorylation (Crowtheret al., 1994; Schneider et al., 1999). Hyperphosphorylation of tau atmany sites appears to precede assembly into filaments, based on findingsin mouse lines expressing human tau with FTDP-17T mutations (Lewis etal. 2000; Allen et al. 2002). Many in vitro studies taken togethersuggest (a) that the microtubule binding domain is important forassembly of tau filaments; and (b) that formation of tau filamentsrequires conformational change(s) of tau. These studies also indicatethat none of tau modifications described therein are alone capable toinduce filamentous tau formations that correlate with clinicalexpression of Alzheimer's disease.

Asuni et al., “Immunotherapy Targeting Pathological Tau Conformers in aTangle Mouse Model Reduces Brain Pathology with Associated FunctionalImprovements”, Journal of Neuroscience, 27 (34): 9115-9129 (Aug. 2,2007) discussed a study in which they sought to determine theeffectiveness of active immunization directed against phosphorylated tauconformers in the CNS by immunizing P301L mice with a phosphorylated tauepitope with subsequent analysis of tau pathology and associatedfunctional impairments. They determined that active immunization with aphosphorylated tau epitope Tau 379-408 (P-_(Ser396, 404)) reducesaggregated tau in the brain and slows progression of the tangle-relatedbehavioral pheno-type in the mice.

Gamblin et al. “Caspase Cleavage of Tau: Linking Amyloid andNeurofibrillary Tangles of Alzheimer's Disease”, PNAS Vol 100, No. 17,pp. 10032-10037 (Aug. 19, 2003), reported that tau is proteolized bymultiple caspases at a highly conserved aspartate residue (Asp⁴²¹) inits C terminus in vitro and in neurons treated with amyloid-β (Aβ₁₋₄₂)peptide. Tau was reported to be rapidly cleaved at Asp⁴²¹ in AB-treatedneurons (within 2 hours), and its proteolysis appears to precede thenuclear events of apoptosis. Gamblin et al. also demonstrated thatcaspase cleavage of tau generates a truncated protein that lacks itsC-terminal 20 amino acids and assembles more rapidly and moreextensively into tau filaments in vitro than wild-type tau. Using amonoclonal antibody that specifically recognizes tau truncated atAsp⁴²¹, Gamblin et al. showed that tau is proteolytically cleaved atthis site in the fibrillar pathologies of AD brain, and suggested thatAβ peptides promote pathological tau filament assembly in neurons bytriggering caspase cleavage of tau and generating a proteolytic productwith enhanced polymerization kinetics.

Delobel et al., “Analysis of Tau Phosphorylation and Truncation in aMouse Model of Human Tauopathy”, American Journal of Pathology, Vol.172, No. 1, pp. 123-131 (January 2008), investigated the time course ofthe appearance of phosphorylated and truncated tau in the brain andspinal cord of mice transgenic for human P301S tau protein. Theyreported that soluble tau was strongly phosphorylated at 1 to 6 months,and low levels of phosphorylated, sarkosyl-insoluble tau were detectedat 2 months with a steady increase up to 6 months of age. They furtherreported that tau truncated at D421 was detected at low levels inTris-soluble and detergent soluble tau at 3-6 months of age. Theyconcluded that the late appearance and low abundance of tau ending atD421 indicates that it is unlikely that truncation at this site isnecessary for the assembly of tau into filaments.

Zhang et al., “Truncated Tau at D421 is Associated withNeurodegeneration and Tangle Formation in the Brain of AlzheimerTransgenic Models”, Acta Neuropathol 117:687-697 (2009), analyzedspatial relationships among tau truncation, tau phosphorylation andneurodegeneration or tangle formation in a tau P302L mice and in atriple transgenic mouse model that produces both amyloid plaques andneurofibrillary tangles. They reported that a few neurons were detectedthat contained abundant truncated tau but were lackinghyperphosphorylation, and these neurons exhibited nuclear condensation,while truncated tau was commonly associated with high immunoreactivityof hyperphosphorylated tau and dense Gallyas silver staining. Theyconcluded that tau truncation appears after tau hyperphosphyorylation inthe brain of these two transgenic mouse models, and that accumulation oftruncated tau, in the absence or the presence of physphorylated tau, isclosely associated with a subset of neurons undergoing degeneration orcontaining neurofibrillary tangles.

Likewise, Khurana, et al. (2010), “Lysosomal Dysfunction PromotesCleavage and Neurotoxicity of Tau InVivo”, PLoS Genet 6(7): e1001026.doi:10.1371/journal.pgen.1001026, demonstrated that removing cathepsin Din adult postmitotic neurons leads to aberrant lysosomal expansion andcaspase activation in vivo, suggesting a mechanism for C-terminaltruncation of tau. They concluded that caspase cleavage of tau may be amolecular mechanism through which lysosomal dysfunction andneurodegeneration are causally linked in AD.

Sigurdsson, “Tau-Focused Immunotherapy for Alzheimer's Disease andRelated Tauopathies”, Current Alzheimer Research, Vo. 6, pp. 446-450(2009), immunized transgenic mice expressing the P301L tau mutation witha 30 amino acid tau fragment that contained two phosphorylation sitesthat are prominent in AD (Tau 379-408[P-Ser396,404] and found thatactive immunization targeting this AD phospho-tau epitope reducesaggregated tau in the brain and prevents/slows progression of thetangle-related behavioral phenotype, including cognitive impairment. Heconcluded that these antibodies enter the brain and bind to pathologicaltau within neurons although the therapeutic effect may be at least inpart due to clearance of extracellular tau that may have biologicaleffects.

Calignon et al., “Caspase Activation Precedes and Leads to Tangles”,Nature Vol. 464/22, pp. 1201-1205 (April 2010), using in vivomultiphoton imaging to observe tangles and activation of executionercaspases in living tau transgenic mice (Tg4510 strain), found thatcaspase activation occurs first, and precedes tangle formation by hoursto days. Based on this data, Calignon et al. proposed that caspaseactivation cleaves tau to initiate tangle formation, then truncated taurecruits normal tau to misfold and form tangles. They further suggestedthat tangles are “off pathway” to acute neuronal death, and that solubletau rather than fibrillar tau may be the critical toxic moietyunderlying neurodegeneration.

Kovacech et al., “Tau Truncation is a Productive PosttranslationalModification of Neurofibrillary Degeneration in Alzheimer's Disease”,Current Alzheimer Research, Vol 7, pp. 708-716 (2010), conclude that twoposttranslational modifications of tau found in AD are assumed to playan inducing role in the neurofibrillary degeneration; truncation andhyperphosphorylation, and that it is impossible to precisely determinethe temporal role of phosphorylation in the development of tau pathologybecause tau mutations are known to alter the conformation of the proteinand lead to its higher and faster phosphorylation in vitro.

Horowitz et al., “Early N-Terminal Changes and Caspase-6 Cleavage of Tauin Alzheimer's Disease”, The Journal of Neuroscience, 24(36), pp.7895-7902 (2004), reported immunohistochemical staining in a cohort of35 cases ranging from noncognitively impaired to early AD with a panelof three N-terminal anti-tau antibodies: Tau-12, 5A6, and 9G3-pY18. Ofthese three, the phosphorylation-independent epitope of 5A6 was theearliest to emerge in the pathological lesions of tau, followed by theappearance of the Tau-12 epitope. It was reported that the unmasking ofthe Tau-12 epitope in more mature 5A6-postive tangles was not correlatedwith tau phosphorylation at tyrosine 18 (9G3-pY18). The extremeN-terminus of tau was lost later in the course of tangle evolution,correlating temporally with the appearance of a C-terminalcaspase-truncated epitope lacking residues 422-441. In addition,caspase-6 cleaved the N terminus of tau in vitro, preventingimmunoreactivity with both Tau-12 and 5A6, with the in vitro caspase-6truncation site being identified as Asp13. The authors concluded thattheir results suggested a role for caspase-6 and N-terminal truncationof tau during neurofibrillary tangle evolution and the progression ofAD.

It would be desirable to provide treatments which could interfere in theinitiation of tau changes leading to filament formation or which couldinterfere with filament formation leading to tangles in diseaseconditions such as AD, and to develop therapeutic agents and dosageforms to treat, prevent or interfere in the progression of tauopathies.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

An object of the present invention is therefore to provide methods oftreatment, therapeutic agents and compositions for therapeuticintervention in and/or prevention of Alzheimer's disease and othertauopathies.

It is a further object of the invention to provide antibodies capable ofselectively recognizing a tau truncated at its C-terminus (e.g., at theglutamic acid residue Glu391 or at the aspartic acid residue Asp421) orits N-terminus (e.g., at amino acid Asp13) (e.g., tau1-13, tau14-441,tau14-391, tau391-414, tau1-391, tau1-421, tau14-421, tau14-410,tau391-410, tau14-412, tau391-412, tau 14-383, tau14-381, or tau 14-355,or a fragment of any of the foregoing). These antibodies would onlyrecognize, bind or show reactivity with truncated tau, but will notrecognize, bind or show reactivity with a normal tau protein (e.g., afull length untruncated htau40). In other words, these antibodies wouldrecognize the neoepitope created by cleavage of tau (i.e., the aminoacid sequences of the free N-terminus or the free C-terminus of thepeptide created by cleavage of tau), but will not recognize the samesequence of amino acids present in the normal tau protein. Theseantibodies are therefore not expected to affect the biological functionsof the normal tau protein, and, in the preferred embodiments, areexpected to clear the peptides created by cleavage of tau and minimizeor prevent the neurofiblary tangles formation. These antibodies maytherefore be used in the treatment and/or prevention of AD and othertauopathies and in the preparation of pharmaceutical compositions (e.g.,vaccines) for the treatment and prevention of these disorders.

It is an additional object of the invention to provide antibodiescapable of selectively recognizing an abnormally phosphorylatedtruncated tau, preferably, tau1-13, tau 14-441, tau14-391, tau391-414,tau1-391, tau1-421, tau14-421, tau14-410, tau391-410, tau14-412,tau391-412, tau 14-383, tau14-381, or tau 14-355, or a fragment of anyof the foregoing (e.g., tau phosphorylated at one or more of thefollowing locations: Ser199, Ser202, Ser214, Ser235, Ser396, Ser404,Thr205, Thr231, and Thr212). These antibodies would only recognize, bindor show reactivity with the abnormally phosphorylated truncated tau, butwill not recognize, bind or show reactivity with a normal tau protein(i.e., a full length untruncated htau40). In other words, theseantibodies would recognize the neoepitope created by the cleavage andabnormal phosphorylation of tau, but would not recognize the same butnot phosphorylated sequence of amino acids which is present internallyin the normal tau protein. These antibodies are also not expected toaffect the biological functions of the normal tau protein and/or inhibitcaspase cleavage of tau, and, in the preferred embodiments, are expectedto clear the peptides created by cleavage of tau and minimize or preventthe neurofiblary tangles formation, and are used to treat and/or preventAD and other tauopathies.

In preferred embodiments, antibodies useful in the present inventionshould be suitable for (i) inhibition, reduction, clearance andelimination of tau truncated at its C-terminus, e.g., at the glutamicacid residue Glu391 or at the aspartic acid residue Asp421, or itsN-terminus (e.g., at the aspartic acid residue Asp13), (ii) inhibition,reduction, clearance and elimination of abnormal phosphorylatedtruncated tau (e.g., tau phosphorylated at Ser396 and/or Ser404), and/or(iii) suitable for the prevention of the neurofiblary tangles formationand/or increased clearance of the neurofiblary tangles, all withoutaffecting the biological functions of the normal tau protein. Theseantibodies should therefore be suitable for symptomatic treatment andprevention of Alzheimer's disease and other tauopathies and/or for thepreparation of a pharmaceutical composition for the treatment of thesedisorders.

It is a further object of the invention is to provide an isolatedimmunogenic peptide comprising or consisting of an amino acid sequencewhich is identical to the amino acid sequence of the neoepitope createdby cleavage of tau, e.g., at the glutamic acid residue Glu391, at theaspartic acid residue Asp421, or at the aspartic acid residue Asp13, ora fragment of such peptide, which may be used for inducing animmunogenic response in a mammal, and, in the preferred embodiments, isfor use in the treatment and/or prevention of Alzheimer's disease andother tauopathies and/or for the preparation of a pharmaceuticalcomposition for the treatment of these disorders.

It is a further object of the invention to provide a mimotope comprisingtwo peptides fused together with or without spacer residues, the firstpeptide mimicking the structure of the neoepitope created by cleavage oftau (i.e., the amino acid sequences bound to the free N- or C-terminusportions of a peptide created by cleavage of tau) in a mammal, and thesecond peptide mimicking the structure of a T cell epitope derived froma different source (e.g., tetanus toxoid), which mimotope is suitablefor inducing an immune response in a mammal, and, in the preferredembodiments, is for use in the treatment and/or prevention of AD andother tauopathies and/or in the preparation of a pharmaceuticalcomposition for the treatment of these disorders.

These objects are addressed with the present invention which relates inone preferred aspect to a method of treating or preventing or slowingthe progression of a tangle-related behavioral phenotype in a subject,comprising administering to a subject in need of therapy for Alzheimer'sdisease or other tauopathies of one or more antibodies with aspecificity to abnormal forms of soluble truncated tau protein which isor is potentially neurotoxic, said antibody showing no binding and/orreactivity with a normal tau protein. These antibodies are preferablyspecific for the neoepitope created by cleavage of tau, do not recognizethe same sequence of amino acids present internally in the normal tauprotein and are administered under conditions and in an amount(s)effective to slow, inhibit and/or reverse a tangle-related behavioralphenotype in the subject. In certain preferred embodiments, theantibodies have specificity to a tau truncated at its C-terminus at theglutamic acid residue Glu391 or at the aspartic acid residue Asp421,and/or a tau truncated at its N-terminus at the aspartic acid residueAsp13 (e.g., tau1-13, tau 14-441, tau14-391, tau391-414, tau1-391,tau1-421, tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau14-383, tau14-381, or tau 14-355, or a fragment of any of the foregoing)and are administered under conditions and in an amount(s) effective toslow, inhibit and/or reverse a tangle-related behavioral phenotype inthe subject. In certain embodiments, these antibodies selectivelyrecognize a peptide comprising or consisting an amino acid sequence ofamino acids 2-30, or a fragment thereof, of tau; a peptide comprising orconsisting of an amino acid sequence of amino acids 380-405, or afragment thereof, of tau; and/or a peptide comprising or consisting ofan amino acid sequence comprising or consisting of amino acids 410-436,or a fragment thereof, of tau; which peptide(s) is(are) created bycleavage of tau; and do not recognize these sequence when thesesequences are present internally in the uncleaved/untruncated tau. Insome of these embodiments, these antibodies selectively recognize aC-terminal of tau1-421 (ΔTau), e.g., amino acid sequences comprising orconsisting of tau416-421, tau417-421, tau418-421, or tau419-421 of ΔTau,do not recognize these sequences in htau40, and do not inhibit caspasecleavage of tau (e.g., at Asp421).

The invention is also related to the administration to a subject in needof therapy for Alzheimer's disease or other tauopathies, of one or moreantibodies with a specificity to abnormal forms of tau protein which areconformationally different from normal tau and/or specificity totruncated tau, said antibodies showing no binding and/or reactivity witha normal tau protein. These antibodies are preferably specific for theneoepitope created by cleavage of tau, and do not recognize the samesequence of amino acids when present internally in the normal tauprotein. In certain preferred embodiments, the antibodies havespecificity to a tau truncated at its C-terminus at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, and/or a tautruncated at its N-terminus at the aspartic acid residue Asp13 (e.g.,tau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau 14-383,tau14-381, or tau 14-355, or a fragment of any of the foregoing), andare administered under conditions and in an amount(s) effective toprevent aggregation, inhibit aggregation and/or promote clearance ofaggregates from the brain of a subject. In some of these embodiments,these antibodies selectively recognize a C-terminal of tau1-421 (ΔTau),e.g., amino acid sequences comprising or consisting of tau416-421,tau417-421, tau418-421, or tau419-421 of ΔTau, do not recognize thesesequences in htau40, and do not inhibit caspase cleavage of tau (e.g.,at Asp421).

Another aspect of the present invention includes a method of slowingprogression of a tangle-related behavioral phenotype in a subject. Thismethod includes administration to a subject in need of therapy forAlzheimer's disease or other tauopathies, of one or more antibodies withspecificity to abnormal forms of tau protein (e.g., truncated tau) whichare linear or conformationally different from normal tau, saidantibodies showing no binding and/or reactivity with a normal tauprotein. These antibodies are preferably specific for the neoepitopecreated by cleavage of tau and do not recognize the same sequence ofamino acids present internally in the normal tau protein. Theseantibodies are therefore not expected to affect the biological functionsof the normal tau protein, and, in the preferred embodiments, areexpected to clear the peptides created by cleavage of tau and minimizeor prevent the neurofiblary tangles formation. In certain preferredembodiments, the antibodies have specificity to a tau truncated at itsC-terminus at the glutamic acid residue Glu391 or at the aspartic acidresidue Asp421, and/or a tau truncated at its N-terminus at the asparticacid residue Asp13 (e.g., tau1-13, tau 14-441, tau14-391, tau391-414,tau1-391, tau1-421, tau14-421, tau14-410, tau391-410, tau14-412,tau391-412, tau 14-383, tau14-381, or tau 14-355, or a fragment of anyof the foregoing), and are administered under conditions and in anamount(s) effective to slow, inhibit and/or reverse a tangle-relatedbehavioral phenotype in the subject.

In another aspect of the present invention, the antibodies administeredselectively recognize a phosphorylated form of the abnormal tau protein(i.e., a truncated tau) and show no binding and/or reactivity with anormal tau protein. These antibodies are therefore not expected toaffect the biological functions of the normal tau protein, and, in thepreferred embodiments, are expected to clear the peptides created bycleavage of tau and minimize or prevent the neurofiblary tanglesformation.

In certain preferred embodiments, the antibodies administeredselectively recognize a phosphorylated form of a tau truncated at itsC-terminus at the glutamic acid residue Glu391 or at the aspartic acidresidue Asp421, or a tau truncated at its N-terminus at the asparticacid residue Asp13. Preferably, only the linear or conformationallydifferent form of truncated tau protein is selectively recognized by theantibodies of the present invention, and the antibodies show no bindingand/or affinity with the normal tau protein (i.e., shows no reactivityto the normal untruncated htau40). In certain embodiments, theseantibodies recognize a phosphorylated peptide comprising or consistingan amino acid sequence of amino acids 1-30, or a fragment thereof, oftau; a phosphorylated peptide comprising or consisting of an amino acidsequence of amino acids 380-405, or a fragment thereof, of tau; and/or aphosphorylated peptide comprising or consisting of an amino acidsequence of amino acids 410-436, or a fragment thereof, of tau; whichpeptide(s) is(are) created by cleavage of tau; and do not recognizethese sequences when these sequences are present internally in theuncleaved/untruncated tau. In some of these embodiments, theseantibodies selectively recognize a C-terminal of ΔTau, e.g., amino acidsequences comprising or consisting of tau416-421, tau417-421,tau418-421, or tau419-421 of ΔTau, and do not recognize these sequencesin htau40.

The invention is further directed to a pharmaceutical compositioncomprising one or more antibodies with specificity to a tau proteintruncated at its C-terminus and/or N-terminus. In certain embodiments,the antibody selectively recognizes a soluble, pre-tangle tau proteintruncated at the glutamic acid residue Glu391, or at the aspartic acidresidue Asp421, or at its N-terminus at the aspartic acid residue Asp13.In preferred embodiments, the antibodies show no binding and/or affinitywith the normal tau protein (i.e., show no reactivity to the normaluntruncated tau protein). In some of these embodiments, the antibody isspecific for ΔTau, and shows no binding and/or affinity to htau40. Inthe preferred embodiments, the composition is for the treatment ofAlzheimer's disease.

The invention is further directed to a pharmaceutical composition forthe treatment and/or prevention of Alzheimer's disease, the compositioncomprising a plurality of antibodies which are specific for theneoepitope created by cleavage of tau (i.e., the amino acid sequencesbound to the free N- or C-terminus portions of a peptide created bycleavage of tau), and do not recognize the same sequence of amino acidswhen present internally in the normal tau protein. In the preferredembodiments, the neoepitope comprises or consists of a sequence selectedfrom SEQ ID No: 7-94 or 116, or a fragment thereof.

The invention is further directed to antibodies that recognize eitherlinear or conformational free-end epitopes of truncated tau. Inpreferred embodiments, the antibodies show no binding and/or affinitywith the normal tau protein (i.e., shows no reactivity to the normaluntruncated tau protein).

The invention is also directed in part to an immunogenic peptide (e.g.,an isolated immunogenic peptide), comprising a portion or fragment of atruncated tau, e.g., expressed by a virus or bacteria, incorporated intoa genome or episome of the virus or bacteria (i.e., the virus orbacteria comprises a gene encoding for the immunogenic peptide),isolated from a mammal, synthesized chemically, or produced usingrecombinant DNA techniques, as part of an immunogenic composition. Inall of these embodiments, the immunogenic portion of the peptidescomprises a linear sequence of two, three, four, five, six, seven,eight, nine, or ten amino acids covalently bound to a free N-terminus ora free C-terminus of a truncated tau, which sequence is identical to thesequence of the first two, three, four, five, six, seven, eight, nine orten amino acids or the last two, three, four, five, six, seven, eight,nine or ten amino acids of a peptide (e.g., ΔTau) created by cleavage oftau. In certain preferred embodiments, the immunogenic peptide comprisesa portion of a tau protein truncated at its C-terminus at the glutamicacid residue Glu391, a portion of the tau protein truncated at itsC-terminus at the aspartic acid residue Asp421, a tau truncated at itsN-terminus at the aspartic acid residue Asp13, or combinations thereof(e.g., tau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau 14-383,tau14-381, or tau 14-355, or a fragment of any of the foregoing). Theimmunogenic portion of the peptide, in the preferred embodiments,comprises or consists of an amino acid sequence which is selected fromSEQ ID Nos: 7-94 or 116, or a fragment thereof. The immunogenic peptideis capable of inducing an immunogenic response in a mammal, and,preferably, is for use in the treatment and/or prevention of Alzheimer'sdisease and other tauopathies and/or in the preparation of apharmaceutical composition for the treatment of these disorders. In thepreferred embodiments, the immunogenic response is the production of theneoepitope-specific antibodies described herein, e.g., in-situ of aliving mammal (e.g., human).

The invention is also directed to a mimotope comprising two peptidesfused together with or without spacer residues, the first peptidemimicking the structure of the neoepitope created by cleavage of tau(i.e., the amino acid sequences bound to the free N- or C-terminusportions of a peptide created by cleavage of tau) in a mammal, and thesecond peptide mimicking the structure of a T cell epitope derived froma different source (e.g., tetanus toxoid), which mimotope is suitablefor inducing an immune response in a mammal. In the preferredembodiments, the immunogenic response is the production of theneoepitope-specific antibodies described herein. In the preferredembodiments, is for use in the treatment and/or prevention ofAlzheimer's disease and other tauopathies and/or in the preparation of apharmaceutical composition for the treatment of these disorders.

The invention is also directed to a pharmaceutical compositioncomprising a mimotope comprising two peptides fused together with orwithout spacer residues, the first peptide mimicking the structure ofthe neoepitope created by cleavage of tau (i.e., the amino acidsequences bound to the free N- or C-terminus portions of a peptidecreated by cleavage of tau) in a mammal, and the second peptidemimicking the structure of a T cell epitope derived from a differentsource (e.g., tetanus toxoid), which mimotope is suitable for inducingan immune response in a mammal. In the preferred embodiments, theimmunogenic response is the production of the neoepitope-specificantibodies described herein. The composition is used for inducing animmunogenic response in a mammal, and, in the preferred embodiments, forthe treatment and/or prevention of Alzheimer's disease and othertauopathies and/or in the preparation of a pharmaceutical compositionfor the treatment of these disorders.

The invention is further directed to a pharmaceutical compositioncomprising (i) a mimotope comprising two peptides fused together with orwithout spacer residues, the first peptide mimicking the structure ofthe neoepitope created by cleavage of tau (i.e., the amino acidsequences bound to the free N- or C-terminus portions of a peptidecreated by cleavage of tau) in a mammal, and the second peptidemimicking the structure of a T cell epitope derived from a differentsource (e.g., tetanus toxoid); (ii) a mimotope comprising two peptidesfused together with or without spacer residues, the first peptidemimicking the structure of the neoepitope created by cleavage of APP(i.e., the amino acid sequences bound to the free N- or C-terminusportions of a peptide created by cleavage of APP) in a mammal, and thesecond peptide mimicking the structure of a T cell epitope derived froma different source (e.g., tetanus toxoid). The composition is used forinducing an immunogenic response in a mammal, and in the preferredembodiments, is for use in the treatment and/or prevention ofAlzheimer's disease and other tauopathies and/or in the preparation of apharmaceutical composition for the treatment of these disorders.

The invention is further directed to a method of treating, preventing,and/or slowing progression of a tangle-related behavioral phenotype in asubject, comprising administering to a subject in need of such treatmentone or more free end-specific antibodies generated from syntheticpeptides comprising immunogenic linear or conformational sequences ofabnormal tau, the antibodies that selectively recognize free ends oftruncated tau (e.g., soluble truncated tau), and show no reactivity(binding or affinity to normal untruncated tau). In certain embodiments,these antibodies may (i) inhibit or slow down, e.g., tau polymerizationand formation of neurofibrillary tangles, and (ii) promote clearance ofabnormal tau and/or the agents responsible for formation of abnormaltau.

Another aspect of the present invention includes a method of preventingor treating Alzheimer's Disease or other tauopathies in a subject, viathe administration of a truncated tau protein, preferably a tautruncated at its C-terminus at the glutamic acid residue Glu391 or atthe aspartic acid residue Asp421 (e.g., ΔTau), and/or a tau truncated atits N-terminus at the aspartic acid residue Asp13, or a fragment of anyof the foregoing, under conditions and in the amounts to induce in-situproduction of free end-specific antibodies to the truncated protein(s)and effective to prevent or treat Alzheimer's Disease or othertauopathies. In the preferred embodiments, the free end-specificantibodies selectively recognize a peptide comprising or consisting anamino acid sequence of amino acids 2-30, or a fragment thereof, of tau;a peptide comprising or consisting of an amino acid sequence of aminoacids 380-405, or a fragment thereof, of tau; and/or a peptidecomprising or consisting of an amino acid sequence of amino acids410-436, or a fragment thereof, of tau; which peptide(s) is(are) createdby cleavage of tau; but do not recognize these sequence when thesesequences are present internally in the uncleaved/untruncated tau. Insome of these embodiments, the method comprises administration of ΔTau,or a fragment thereof, and the antibodies produced in response to thisadministration selectively recognize a C-terminal of ΔTau, and do notrecognize these sequences in htau40.

Another aspect of the present invention includes a method of slowingprogression of a tangle-related behavioral phenotype in a subject. Thismethod includes administration to a subject in need of therapy forAlzheimer's disease or other tauopathies, of a truncated tau protein,preferably a tau truncated at its C-terminus at glutamic acid residueGlu391 or at the aspartic acid residue Asp421, and/or a tau truncated atits N-terminus at the aspartic acid residue Asp13, under conditions andin the amounts to induce in situ production of free end-specificantibodies to the truncated protein(s) and effective to slow, inhibitand/or reverse a tangle-related behavioral phenotype in a subject.

The invention is further directed in part to a gene therapy vectoroperably linked to a gene encoding for an immunogen, the immunogencomprising a portion of a truncated tau protein. In certain embodiments,the truncated tau protein is selected from the group consisting of a tauprotein truncated at its C-terminus at the glutamic acid residue Glu391,a tau protein truncated at the aspartic acid residue Asp421, a tautruncated at its N-terminus at the aspartic acid residue Asp13, andcombinations thereof (e.g., tau1-13, tau 14-441, tau14-391, tau391-414,tau1-391, tau1-421, tau14-421, tau14-410, tau391-410, tau14-412,tau391-412, tau 14-383, tau14-381, or tau 14-355, or a fragment of anyof the foregoing). In some of these embodiments, the immunogen comprisesor consists of the last ten, nine, eight, seven, six, five, four orthree amino acids of ΔTau.

The invention is also directed in part to a pharmaceutical compositioncomprising naked DNA encoding an immunogen comprising a portion of theprotein selected from the group consisting of a tau protein truncated atits C-terminus at the glutamic acid residue Glu391, a tau proteintruncated at the aspartic acid residue Asp421, a tau truncated at itsN-terminus at the aspartic acid residue Asp13, and combinations thereof(e.g., tau1-13, tau 14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau 14-383,tau14-381, or tau 14-355, or a fragment of any of the foregoing, andcombinations thereof).

The invention is further directed to a vaccine which comprises a portionof one or more abnormal or truncated tau protein(s) as set forth hereinin combination with a pharmaceutically acceptable carrier, or a vaccinewhich comprises one or more antibodies specific for a portion(s) of anabnormal or truncated tau protein(s) as set forth herein and capable ofcrossing the blood brain barrier in combination with a pharmaceuticallyacceptable carrier. In the preferred embodiments, the vaccine is for thetreatment of Alzheimer's disease, and comprises a mimotope fused with abacterial peptide, the mimotope mimicking the structure of theneoepitope created by cleavage of tau (i.e., the amino acid sequencesbound to the free N- or C-terminus portions of a peptide created bycleavage of tau) in a mammal, and the bacterial peptide comprising orconsisting of a natural bacterial tetanus toxoid or equivalent. The useof the mimotope, in the preferred embodiments, prevents a possibility ofan autoimmune response which does not apply to a bacterial peptide. Thevaccine may or may not comprise an additional mimitope comprising amimotope mimicking the structure of the neoepitope created by cleavageof APP (i.e., the amino acid sequences bound to the free N- orC-terminus portions of a peptide created by cleavage of APP) in amammal, the additional mimitope fused, with or without spacer residues,to a bacterial peptide which is a natural bacterial tetanus toxoid orequivalent. The vaccine is for inducing an immunogenic response in amammal. In the preferred embodiments, the immunogenic response is theproduction of the neoepitope-specific antibodies described herein. Inthe preferred embodiments, the vaccine is for use in the treatmentand/or prevention of Alzheimer's disease and other tauopathies and/or inthe preparation of a pharmaceutical composition for the treatment ofthese disorders.

The invention is further directed to a vaccine which comprises amimotope mimicking the structure of the neoepitope created by cleavageof tau in a mammal, the mimotope fused, with or without spacer residues,to a bacterial peptide comprising or consisting of a natural bacterialtetanus toxoid or equivalent, wherein the neoepitope comprises orconsists of an amino acid sequence of amino acids 1-30, or a fragmentthereof, of tau; a peptide comprising or consisting of an amino acidsequence of amino acids 380-405, or a fragment thereof, of tau; and/or apeptide comprising or consisting of an amino acid sequence of aminoacids 410-436, or a fragment thereof, of tau; and the mimitope issuitable for inducing an immunogenic response in a mammal. In some ofthese embodiments, the neoepitope comprises or consists of amino acids16-421, 17-421, 18-421, or 19-421 of ΔTau. In the preferred embodiments,the vaccine is for use in a pharmaceutical composition for the treatmentand/or prevention of Alzheimer's disease and other tauopathies.

The invention is further directed to a vaccine which comprises (i) amimotope mimicking the structure of the neoepitope created by cleavageof tau in a mammal (e.g., at Asp421), the mimotope fused, with orwithout spacer residues, to a bacterial peptide comprising or consistinga structure of a T cell epitope derived from a different source (e.g.,tetanus toxoid); and (ii) a mimitope mimicking the structure of theneoepitope created by cleavage of Aβ in a mammal, fused, with or withoutspacer residues, to a bacterial peptide comprising or consisting thestructure of a T cell epitope derived from a different source (e.g.,tetanus toxoid). The T cell epitope in the first mimotope and the secondmimotope may be the same or different. In certain embodiments, the Tcell epitope in the first mimotope and in the second mimotope comprisethe same structure as a well-studied tetanus toxoid promiscuous epitopeof SEQ ID No: 95 (Ho et al., 1990; Panina-Bordignon et al. 1989), asthis epitope is known to work in a number of diverse human geneticbackgrounds (Valmori et al., 1992 and 1994). In the preferredembodiments, the vaccine is for the treatment and/or prevention ofAlzheimer's disease and other tauopathies.

In certain embodiments, the invention is directed to a pharmaceuticalcomposition comprising a chimeric peptide(s) comprising (i) a 2-10 or2-6 amino acid residue from the free N- or C-terminus of a truncated tau(e.g., ΔTau) fused together with or without a spacer to (ii) apromiscuous T helper cell epitope derived from a different source thanthe amino acid residue. The truncated tau is selected from the groupconsisting of tau truncated at its C-terminus at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, or a tautruncated at its N-terminus, e.g., at the aspartic acid residue Asp13.In certain embodiments, the truncated tau is ΔTau. In certainembodiments, the T helper cell epitope is the well-studied tetanustoxoid promiscuous epitope of SEQ ID No: 95. The composition comprisingan immunizing effective amount of the chimeric peptide or peptides and apharmaceutically acceptable carrier, excipient, diluent, or auxiliaryagent may then be administered to a mammal (e.g., human) to induceformation of antibodies which are specific for the neoepitope created bycleavage of tau (i.e., the amino acid sequences bound to the free N- orC-terminus portions of a peptide created by cleavage of tau), and do notrecognize the same sequence of amino acids when present in the normaltau protein.

The promiscuous T helper cell epitope may be a T cell epitope derivedfrom, e.g., tetanus toxin, pertussis toxin, diphtheria toxin, measlesvirus F protein, hepatitis B virus surface antigen, Chlamydiatrachomitis major outer membrane protein, Plasmodium falciparumcircumsporozoite, Schistosoma mansoni triose phosphate isomerase, orEscherichia coli TraT. In the preferred embodiments the T cell epitopeis a well-studied tetanus toxoid promiscuous epitope of SEQ ID No: 95(Ho et al., 1990; Panina-Bordignon et al. 1989), as this epitope isknown to work in a number of diverse human genetic backgrounds (Valmoriet al., 1992 and 1994).

Further aspects of the invention are directed to a truncated tau proteinwherein the last 20 amino acids at the C-terminal or N-terminal of tauare taken off and are not present in the truncated protein (e.g., ΔTau);the immunogenic portion of the truncated tau protein; genes encoding fortruncated protein and/or peptide containing the immunogenic portion ofthe protein; antibodies selective/specific for the truncatedprotein-monoclonal, polyclonal, chimeric, recombinant, humanized, andportions of any of the foregoing; produced in situ and ex situ;transgenic “animals” secreting antibodies selective/specific for thetruncated protein; active immunization (administration of truncatedprotein or immunogenic portions thereof to a subject); passiveimmunization (administration of antibodies in accordance with theinvention to a subject); and pharmaceutical formulations for active andpassive immunizations.

The immunogenic fragment of the truncated tau, in certain embodiments,comprises a linear sequence of two, three, four, five, six or sevenamino acids covalently bound to a free N-terminus or a free C-terminus,which sequence is identical to the sequence of the first two, three,four, five, six or seven amino acids or the last two, three, four, five,six or seven amino acids of a peptide (e.g., ΔTau) created by cleavageof tau. In certain embodiments, the peptide comprises or consists of anamino acid sequence of amino acids 1-30, or a fragment thereof, of tau;an amino acid sequence of amino acids 380-405, or a fragment thereof, oftau; or an amino acid sequence of amino acids 410-436, or a fragmentthereof. In some of these embodiments, the sequence comprises orconsists of amino acids 16-421, 17-421, 18-421, or 19-421 of ΔTau.

In one embodiment, the immunogenic fragment of the truncated taucomprises a linear sequence of at least five amino acids of the tauprotein which ensures that the specific free amino group at theN-terminus constitutes an essential part of the epitope recognized bythe new linear epitope free-end specific antibody. In other embodiments,fragments of truncated tau have at least the last 20, 30 or 45 aminoacids of the truncated tau, and linear epitope free-end specific orconformation-specific antibodies are generated. In certain furtherpreferred embodiments, the invention is directed to a vaccine which is acombination of a composition providing immunization against cleavageproducts of tau (i.e., truncated tau protein) and a compositionproviding immunization against cleavage products of APP.

In certain preferred embodiments, the composition providing immunizationagainst truncated tau protein comprises a chimeric peptide(s) comprising(i) a 2-10 or 2-6 amino acid residue from the free N- or C-terminus of atruncated tau (e.g., ΔTau) fused together with or without a spacer to(ii) a promiscuous T helper cell epitope derived from a different sourcethan the amino acid residue; and the composition providing immunizationagainst cleavage product of APP comprises a chimeric peptide(s)comprising (i) a 2-10 or 2-6 amino acid residue from the free N or Cterminus of a truncated APP (e.g., Aβ₁₋₄₀, Aβ₁₋₄₂, Aβ₁₋₄₃, etc.) fusedwith or without a spacer to (ii) a promiscuous T helper cell epitopederived from a different source than the amino acid residue. The T cellepitope in the composition providing immunization against cleavageproducts of tau and in the composition providing immunization againstcleavage products of APP may be the same or different. In certainembodiments, the T cell epitope of both compositions comprises thewell-studied tetanus toxoid promiscuous epitope of SEQ ID No: 95.

“Antibody” as used herein is meant to include intact molecules andfragments thereof, as well as synthetic and biological derivativesthereof, such as for example Fab, F(ab′₂ an F_(V) fragments-free orexpressed, e.g., on the surface of filamentous phage on pIII or pVIII orother surface proteins, or on the surface of bacteria, which are capableof binding an antigen. Fab, F(ab′₂ and F_(V) fragments lack the F_(C)fragments of intact antibody, clear more rapidly from the circulationand may have less non-specific tissue binding of antibody. FurthermoreF_(V) antibody (often called as minibody) can be easily engineered tocarry on its C-terminus specific tracer and used for early intravitalpresymptomatic diagnosis of AD, since stage I, II and III of AD that isrecognized by the antibodies according to the present invention is notassociated with intellectual decline. The term antibody encompasses,e.g., chimeric and humanized antibodies. The antibody may be amonoclonal antibody or a polyclonal antibody. It also encompassesrecombinant antibodies. The antibodies may preferably be linearantibodies, or conformational antibodies.

The terms “does not bind,” “does not recognize,” and “does not showreactivity” as used in the present application mean either that anantibody shows no detectible binding with a peptide or protein (e.g.,htau40), or that the antibody's equilibrium constant KD with the peptideor protein is from 1×10⁻⁴ molar to 1×10⁻⁶ M, as measured by a surfaceplasmon resonance assay utilizing peptide captured on streptavidin chip.

The terms “binds specifically,” “specifically recognize,” “selectivelyrecognizes,” “having specificity,” and “specific for” as used in thepresent specification mean that an antibody binds the antigen it isspecific for (e.g., the neopitope created by cleavage of htau at Asp421)with equilibrium constant KD of from 1×10⁻⁹M to 1×10⁻¹¹ M, as measuredby a surface plasmon resonance assay utilizing peptide captured onstrepvidin chip; and has an equilibrium constant KD with other peptidesor proteins (e.g., htau40) which is from 1×10⁻⁴M to 1×10⁻⁶ M, asmeasured by the surface plasmon resonance assay utilizing peptidecaptured on strepvidin chip, or shows no detectible binding with theseother peptides or proteins.

The term “tau protein” as used in the present application refers to theany one of known isoforms of tau (e.g., longest isoform of humanmicrotubule associated protein tau containing all alternatively splicedinserts as described in M. Goedert et al., 1989 (htau40)).

The term “humanized antibody” is referred herein above to an antibody inwhich the complementary-determining regions (CDRs) of a mouse or othernon-human antibody are grafted onto a human antibody framework. By humanantibody framework is meant the entire human antibody excluding theCDRs.

The term “chimeric antibody” refers to an antibody in which the whole ofthe variable regions of a mouse or rat antibody are expressed along withhuman constant regions.

The term “treating” is referred hereinabove to delay or prevent theonset slow the progression or ameliorate the symptoms related toAlzheimer's disease or other disease or disorder characterized by Aβdeposition.

The term “mimotope” as used in the present application is amacromolecule, often a peptide (i.e., an immunogenic peptide orimmunogen), which mimics the structure of an epitope.

The term “tauopathy” refers to tau-related disorders or conditions,e.g., Alzheimer's Disease, Progressive Supranuclear Palsy (PSP),Corticobasal Degeneration (CBD), Pick's Disease, Frontotemporal dementiaand Parkinsonism associated with chromosome 17 (FTDP-17), Parkinson'sdisease, stroke, traumatic brain injury, mild cognitive impairment andthe like.

The terms “immunogen” refers to a molecule capable of being bound by anantibody, a B cell receptor (BCR), or a T cell receptor (TCR) ifpresented by MHC molecules. The term “immunogen”, as used herein, alsoencompasses T-cell epitopes. An immunogen can additionally be capable ofbeing recognized by the immune system and/or being capable of inducing ahumoral immune response and/or cellular immune response leading to theactivation of B- and/or T-lymphocytes. This may, however, require that,at least in certain cases, the immunogen contains or is linked to a Thelper cell epitope and is given an adjuvant. An immunogen can have oneor more epitopes (e.g., B- and T-epitopes). The “immunogen” as usedherein may also be mixtures of several individual immunogens. The term“immunogen” encompasses, but are not limited to, peptides.

As used herein, the term “phosphorylated” in reference to an amino acidresidue refers to the presence of a phosphate group on the side chain ofthe residue where a hydroxyl group is otherwise normally present. Suchphosphorylation typically occurs as a substitution of the hydrogen atomfrom a hydroxyl group for a phosphate group (—PO₃H₂). As recognized bythose of skill in the art, depending on the pH of the local environment,this phosphate group can exist as an uncharged, neutral group (—PO₃H₂),or with a single (—PO₃H⁻), or double (—PO₃ ²⁻) negative charge. Aminoacid residues that can typically be phosphorylated include the sidechains of serine, threonine, and tyrosine.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular unless the content clearly dictates otherwise.

The term “isolated” with respect to an immunogenic peptide refer to apeptide that by virtue of its origin or source of derivation (1) is notassociated with naturally associated components that accompany it in itsnative state, (2) is substantially free of other proteins from the samespecies, (3) is expressed by a cell from a different species, or (4)does not occur in nature. Thus, a peptide that is chemically synthesizedor synthesized in a cellular system different from the cell from whichit naturally originates will be “isolated” from its naturally associatedcomponents. A peptide may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “neoepitope” as used in the present application refers to anon-naturally occurring epitope created as a result of cleavage of apre-cursor protein (e.g., tau, APP, etc.) and/or phosphorylated Tau.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mechanism for the formation of neurofiblary tangles.

DETAILED DESCRIPTION OF THE INVENTION

A proposed model for the formation of neurofiblary tangles stipulatesthat apoptotic stimuli (e.g., Aβ₁₋₄₂) results in caspase-cleavage oftau, e.g., at amino acid Asp421, which leads to increased pathologicalformation of neurofibrillary filaments (NTFs) and paired helicalfilaments (PHFs), as well as pathological tau aggregation. For example,Rissman et al., J. Clin. Invest. 114: 121-130 (2004), propose themechanism for the formation of neurofiblary tangles shown in FIG. 1.According to Rissman et al., exposure to apoptotic stimuli such asAβ₁₋₄₂ result in caspase-cleavage of tau after Asp421 (I);caspase-cleaved tau rapidly adopts the MCI conformational epitope (II),which leads to increased filament formation and tau aggregation (III);to compensate for tau aggregation, tau may subsequently behyperphosphorylated and disassociate from microtubule (IV); and, as aresult, caspase cleavage of tau may lead to PHF formation (V).

Increased formation of pathological NTFs and PHFs and pathological tauaggregation is commonly seen in mammals suffering from progressiveneurodegenerative disorders (e.g., AD and other tauopathies) and isassociated with loss of cognitive and behavioral functions in mammals.Since cleavage of tau by executioner caspases after exposure to anapoptotic stimuli (e.g., Aβ) is thought to result in a form that isespecially prone to tangle formation, an inhibition or decrease of thecaspase-cleaved tau should therefore decrease and/or prevent formationof pathological NTF and PHF and pathological tau aggregation. Inhibitionor decrease of the caspase-cleaved tau should also be useful in thetreatment and/or prevention of the progressive neurodegenerativediseases.

The present invention is directed to antibodies specific for free endsof truncated tau (e.g., caspase-cleaved htau40 (e.g., ΔTau) and showingno binding and/or reactivity with the normal tau and uses of theseantibodies in the treatment and/or prevention of AD and othertauopathies, in clearance of soluble truncated tau from the brain of apatient suffering from AD or another tauopathy, and in preparation ofpharmaceutical compositions for the treatment and/or prevention of thesedisorders. These antibodies would recognize the neoepitope created bycleavage of tau (e.g., C-terminus of ΔTau), but will not recognize thesame sequence of amino acids present in the normal tau protein (e.g.,htau40), which lacks the neoepitope. These antibodies are not expectedto affect the biological functions of the normal tau protein, and areexpected to clear the peptides created by cleavage of tau and minimizeor prevent the pathological NTFs and PHFs formation and pathological tauaggregation. These antibodies are also not expected to inhibit caspasecleavage of htau40 at Asp421.

The antibodies that can selectively recognize the free end(s)(neoepitope(s)) of soluble tau peptides (e.g., ΔTau) formed by thecleavage of tau (e.g., htau40), while not recognizing and showing noreactivity with full length tau protein, are believed to be capable ofdirectly inhibiting polymerization of tau and/or formation of NTFs, PHFsand/or other pathological tau precursors. One significance of usingthese neoepitope-specific antibodies is that these antibodies may beused to clear soluble neurotoxic tau before NTFs and PHFs are formedand/or before tau is pathologically aggregated or polymerized, and/orbefore these neoepitopes become inaccessible or less accessible to theantibodies, and/or the neurological damage is done.

It is specifically contemplated that these antibodies may be used for(i) inhibition, reduction, clearance and elimination of tau truncated atits C-terminus, e.g., at the glutamic acid residue Glu391 or at theaspartic acid residue Asp421, or its N-terminus (e.g., at aspartic acidresidue Asp421), (ii) inhibition, reduction, clearance and eliminationof abnormal phosphorylated truncated tau (e.g., tau phosphorylated atSer396 and/or Ser404), and/or (iii) prevention of NTFs and/or PHFsformation and/or increased clearance of NTFs and PHFs, all withoutaffecting the biological functions of the normal tau protein (e.g.,htau40).

Two neoepitope-specific antibodies specifically contemplated by thepresent invention for the uses described above are: an antibody that isspecific for free C-terminus end of tau truncated at Asp421 (i.e.,ΔTau), and shows no binding and/or reactivity with the normal tau (e.g.,htau40); and an antibody that is specific for free C-terminus end oftau14-421, and shows no binding and/or reactivity with the normal tauprotein. In the preferred embodiments, these antibodies do not inhibitcaspase cleavage of htau40 at Asp421.

It is believed that, as of the effective filing date of the presentapplication, there is no reports in the literature of using antibodiesspecific or selective for soluble pre-tangle truncated tau, e.g., atAsp421, Glu391 and or Asp13 and showing no specific binding orreactivity to full length tau protein (or antibodies havingthree-dimensional structures similar to the antibodies specific forsoluble pre-tangle truncated tau at Asp421, Glu391 and or Asp13) in thetreatment or prevention of AD and other tauopathies. The use of theseneoepitope-specific antibodies for treating AD or another tauopathy isnot obvious, e.g., because the literature does not report where thesoluble truncated forms of tau exist in the cell and if such forms andlocations are accessible to antibodies. This approach also does notdepend on producing conformational antibodies to preformed tau tanglesthat are already causing damage, and is intended to address the problembefore the pathological NTFs and/or PHFs are formed and tau ispathologically aggregated.

The present invention also encompasses a method of treating orpreventing Alzheimer's disease or other tauopathies in a subject. Thismethod includes administering antibodies to truncated tau proteinsselectively recognizing these truncated tau proteins, or portions of thetruncated tau proteins, to a patient under conditions and in the amountseffective to treat or prevent Alzheimer's Disease or other tauopathies.The antibodies may be administered, e.g., intravenously, subcutaneously,nasally, buccally, transdermally, etc., as described in more detailbelow. In certain preferred embodiments, the antibodies selectivelyrecognize soluble pre-tangle truncated tau, and show no binding and/orreactivity with normal tau. The antibodies should therefore facilitateclearance of the truncated tau and should not affect the biologicalfunctions of the normal tau. In certain embodiments, the administeredantibodies block aggregation of ΔTau directly (e.g., by attaching to theC-terminus of ΔTau and, thereby, directly interfering with the abilityof the C-terminus of ΔTau to interact with outer proteins and peptides).The antibodies blocking tau aggregation directly will, preferably, havea low off rate.

In one aspect, the present invention includes a method of promotingclearance of tau aggregates from the brain of a subject. This methodincludes administering antibodies with a specificity to abnormal(truncated) forms of tau protein (or portions of the abnormal(truncated) forms of tau protein) which may or may not beconformationally different from normal tau, the antibodies beingnon-specific for a normal tau protein (show no affinity, binding orreactivity with normal tau), to a mammal (e.g., a human patient). Incertain preferred embodiments, the antibodies have specificity to a tautruncated at its C-terminus at the glutamic acid residue Glu391 or atthe aspartic acid residue Asp421. In other preferred embodiments, theantibodies have specificity to a tau truncated at its N-terminus, e.g.,at amino acid Asp13. In additional preferred embodiments, the antibodieshave specificity to ΔTau (tau1-421) and/or tau14-421. The aggregates tobe cleared include, e.g., neurofibrillary tangles or their pathologicaltau precursors. Neurofibrillary tangles are often associated withneurodegenerative diseases including, for example, Alzheimer's disease,hereditary frontotemporal dementia and parkinsonism linked to chromosome17 (FTDP-17), Pick's disease, sporadic corticobasal degeneration, andprogressive supranuclear palsy. The antibodies may be administered,e.g., intravenously, subcutaneously, nasally, buccally, transdermally,etc., as described in more detail below.

Another aspect of the present invention includes a method of slowing theprogression of or reversing a tangle-related behavioral phenotype in asubject. This method includes administering a truncated tau or a portionof the truncated tau (e.g., as a vaccine), or antibodies specificallyrecognizing a truncated tau or a portion of a truncated tau, underconditions and in the amounts effective to slow or reverse atangle-related behavioral phenotype in a subject. The truncated tau, aportion of truncated tau, or antibodies may be administered, e.g.,intravenously, subcutaneously, nasally, buccally, transdermally, etc.,as described in more detail below.

In certain preferred embodiments, the invention is directed toadministering antibodies with a specificity to abnormal (truncated)forms of tau protein (or portions of the abnormal (truncated) forms oftau protein) which may or may not be conformationally different fromnormal tau, said antibody being non-specific for a normal tau protein(show no binding or reactivity with normal tau), e.g., to a humanpatient. Preferably, these antibodies recognize either linear orconformational free-end epitopes of truncated tau. In certain preferredembodiments, these free end-specific antibodies inhibit taupolymerization. The antibodies may be administered, e.g., intravenously,subcutaneously, nasally, buccally, transdermally, etc., as described inmore detail below.

As stated by Kovacech et al., “Tau Truncation is a ProductivePosttranslational Modification of Neurofibrillary Degeneration inAlzheimer's Disease”, Current Alzheimer Research Vol. 7 pp. 708-716(2010), taking into account that a range of C-terminal tau truncationscan promote tau assembly into paired helical filaments (PHF's), variousN- and C-terminally truncated tau proteins exert abnormal microtubuleassembly, and both Glu391 and Asp421 truncated tau molecules inducedsimilar levels of apoptotic cells, many of the proteins present in thetruncated tau proteome of the diseased brain can serve as inducers oftau neurofibrillary degeneration. Tau mutations are known to alter theconformation of the protein and lead to its higher and fasterphosphorylation in vitro. Truncation of tau can even induce itshyperphosphorylation. While the temporal role of phosphorylation in thedevelopment of tau pathology has not been determined, Kovacech et al.report that the in vivo model of tauopathy based on the truncated tauprotein clearly shows that truncation is a “productive” modificationthat can initiate tau neurofibrillary degeneration. Kovacech et al.further state that immunohistochemical mapping of the distribution ofAsp421 and Glu391 truncated tau in AD brains indicated that theseepitopes appear in a specific temporal order of the tangle development,and propose that the neurofibrillary tangles (NFTs) pass through severalstages during which tau changes conformation several times and becomesprogressively truncated at both N- and C-termini; initially, full lengthtau molecules being assembled in pre-tangle neurons exhibiting theconformational epitope Alz50, and truncation events proposed to ensuesoon after the tangle formation, tau being first truncated at the Asp421cleavage point (e.g., by caspase-3) and later cleaved further at Glu391.

Asuni et al. 2007 reported clearance of tau from the brain usingimmunotherapy. The result was surprising and counterintuitive becausethe target was thought to be mainly intracellular and mainly in thecytoplasm and therefore generally inaccessible to antibodies generatedor delivered outside the cell. Various mechanisms have been postulatedbut none definitively demonstrated to explain how immunotherapy works inthis case. One suggestion was that the tau protein that was cleared fromthe brain of transgenic mice, was in fact extracellular. Anothersuggestion was that the tau-antibody complex formed in a vacuolarcompartment that is linked to the secretory-endosomal pathway. A thirdidea was that antibodies get inside degenerating nerve cells (See reviewby Sigurdsson, Current Alzheimer's Research 2009, 6, 446-450).

Truncated Tau

The abnormal forms of tau proteins which are the subject of the presentinvention typically are truncated tau proteins (e.g. caspase-cleaved tauproteins), most preferably tau truncated at its C-terminus at theglutamic acid residue Glu391 or at the aspartic acid residue Asp421, ora tau truncated at its N-terminus, e.g., at the aspartic acid residueAsp13 (e.g., tau1-13, tau14-441, tau14-391, tau391-414, tau1-391,tau1-421, tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau14-383, tau14-381, or tau14-355, or a fragment of any of the foregoing).In certain embodiments, the truncated tau protein is tau1-421 (ΔTau), ora C-terminal fragment thereof. These abnormal tau proteins may beconformationally different from normal tau and are sometimes referred toas “tauons” (see, e.g., U.S. Patent Publication No. U.S. 2004/0082763,hereby incorporated by reference in its entirety). Differentconformations compared to normal human tau may be attributedpathologically to abnormal truncation at the N-terminus or at theC-terminus or at both termini of the tau molecule.

These abnormal or truncated tau proteins or fragments thereof may beused as immunogens or mimotopes to generate antibodies specific for thetruncated tau protein (e.g., neoepitopes created by cleavage of tau at,e.g., Asp421) and non-specific for untruncated tau, in-situ and ex-situof a subject's brain, and/or administered to a subject to induceformation of the neoepitope-specific antibodies in the subject. Forexample, the above-mentioned truncated tau proteins may be administeredto a mammal (e.g., a human patient) who may be susceptible to theformation of neurofibrillary tangles in order to raise antibodiesagainst such truncated tau proteins if and when they form in vivo. Incertain embodiments, the truncated tau protein comprises an amino acidsequence of amino acids 1-30, or a fragment thereof, of tau; an aminoacid sequence of amino acids 380-405, or a fragment thereof, of tau; oran amino acid sequence of amino acids 410-436, or a fragment thereof, oftau. In certain embodiments, the truncated tau protein is tau1-421(ΔTau), or a C-terminal fragment thereof (e.g., tau411-421, tau412-421,tau413-421, tau414-421, tau 415-421, tau416-421, tau417-421, ortau418-421). In some of these embodiments, the truncated tau protein isphosphorylated at Ser412 and/or Ser413.

In certain embodiments, the truncated tau comprises or consists oftau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421, tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau14-383,tau14-381, tau 14-355, or a fragment of any of the foregoing, of any oneof the six isoforms of the human tau protein. The truncated tau, incertain embodiments, may be phosphorylated at one or more of thefollowing: Ser199, Ser202, Ser214, Ser235, Ser396, Ser404, Thr205,Thr231, and Thr212, if present.

The truncated tau protein described above can be derived from any one ofthe six isoforms of the human tau protein or a segment thereof. Tauprotein has 0, 1, or 2 N-terminal inserts resulting from the splicing ofexons two and three, and either 3 or 4 microtubule-binding domainsresulting from the splicing of exon ten. In certain embodiments, thetruncated tau protein is derived from the longest isoform of tau (i.e.,htau40). The amino acid sequences corresponding to the isoforms of thehuman tau protein of the present invention are given in SEQ ID NOs: 1-6.

SEQ ID NO: 1, the longest tau isoform, htau40, containing two N-terminalinserts and four microtubule binding (2N4R) domains, is as follows:MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 120HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK 180TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 240SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV 300PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 360THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 420DSPQLATLAD EVSASLAKQG L 441SEQ ID NO: 2 contains two N-terminal inserts and three microtubule-binding domains (2N3R) as follows:MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 120HVTQARMVSK SLDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK 180TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 240SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK 300PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAE 360IVYKSPVVSG DTSPAHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL 410SEQ ID NO: 3 contains one N-terminal insert and four microtubule-bindingdomains (IN4R) as follows:MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTGDGSEEPG 60SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSLDGTGSDD KKAKGADGKT 120LIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 180SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 240PGGGKVQIIN KKLDLSNVQS KCGSLDNILH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 300HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 360AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL 412SEQ ID NO: 4 contains zero N-terminal inserts and four microtubule-binding domains (0N4R) as follows:MAEPRQEFEV MEDHAGTYGL GDRLDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 60AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 120PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VATPPKSPSS 180AKSRLQTAPV PMPDLKNVKS LIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK 240HVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVE VKSEKLDFKD RVQSKIGSLD 300NITHVPGGGN KKIETHKLTF RENAKALTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID 360MVDSPQLATL ADEVSASLAK QGL 383SEQ ID NO: 5 contains one N-terminal insert and three microtubule-binding domains (1N3R) as follows:MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 120KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 180SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 240PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 300THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 360DSPQLATLAD EVSASLAKQG L 381SEQ ID NO: 6 contains zero N-terminal inserts and three microtubule-binding domains (0N3R) as follows:MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 60AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 120PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 180AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 240HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIGTHKLTFR ENAKAKTDHG 300AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL 352

The truncated tau protein of the present invention can be phosphorylatedat one or more amino acid residues. In one embodiment, the truncated tauprotein is fully phosphorylated. Amino acid residues in the full lengthtau protein, SEQ ID NO:1, that are or can be phosphorylated includetyrosines at amino acid positions 18, 29, 97, 310, and 394; serines atamino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238,262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; andthreonines at amino acids positions 175, 181, 205, 212, 217, 231, and403. Amino acid residues which are or can be phosphorylated in SEQ IDNO:2 include tyrosines at positions 18, 29, 197, 279, and 363; serinesat positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293,325, 365, 369, 373, 378, 381, 382, 391; and threonine at positions 175,181, 205, 212, 217, 231, 372. Amino acid residues which are or can bephosphorylated in SEQ ID NO:3 include tyrosines at positions 18, 29,168, 281, and 365; serines at positions 155, 156, 169, 170, 173, 179,185, 206, 208, 209, 233, 264, 295, 327, 367, 371, 375, 380, 383, 384,393; and threonines at positions 146, 152, 176, 183, 188, 202, and 374.Amino acid residues which are or can be phosphorylated in SEQ ID NO:4include tyrosines at positions 18, 29, 139, 252, 336; serines atpositions 126, 127, 140, 141, 144, 150, 156, 177, 179, 180, 204, 235,266, 298, 338, 342, 346, 351, 354, 355, 364, and threonines at positions117, 123, 147, 154, 159, 173, and 345. Amino acid residues which are orcan be phosphorylated residues in SEQ ID NO: 5 include tyrosines atpositions 18, 29, 168, 250, 334; serines at positions 155, 156, 169,170, 173, 179, 185, 206, 208, 209, 233, 264, 296, 336, 340, 344, 349,352, 353, 362; and threonines at positions 146, 152, 1376, 183, 188,202, 343. Amino acid residues which are or can be phosphorylated in SEQID NO: 6 include tyrosines at positions 18, 29, 139, 221, and 305;serines at positions 126, 127, 140, 141, 144, 150, 156, 177, 179, 180,204, 235, 267, 307, 311, 315, 320, 323, 324, 333; and threonine atpositions 117, 123, 147, 154, 159, 173, and 314. Additional tyrosine,serine or threonine amino acids within the tau sequences may also bephosphorylated.

Thus, a further aspect of the invention relates to a phosphorylatedtruncated tau protein or a portion thereof and a pharmaceuticalcomposition containing the phosphorylated truncated tau protein or aportion thereof. In certain embodiments, the truncated tau protein istau1-421 (ΔTau), or a C-terminal fragment thereof (e.g., tau411-421,tau412-421, tau413-421, tau414-421, tau 415-421, tau416-421, tau417-421,tau418-421, or tau419-421), which is phosphorylated at one or more ofthe following Tyr¹⁸, Tyr²⁹, Ser¹⁸⁴, Ser¹⁸⁵, Ser¹⁹⁸, Ser¹⁹⁹, Ser²⁰²,Ser²⁰⁸, Ser²¹⁴, Ser²³⁵, Ser²³⁷, Ser²³⁸, Ser²⁶², Ser²⁹³, Thr¹⁷⁵, Thr¹⁸¹,Thr²⁰⁵, Thr²¹², Thr²¹⁷, Ser⁴¹¹, Ser⁴¹², Ser⁴¹⁶, Thr⁴¹⁴. In some of theseembodiments, the truncated tau protein is a C-terminal fragment of ΔTauwhich is phosphorylated at one or more of the following Ser⁴¹¹, Ser⁴¹²,Ser⁴¹⁶, and Thr⁴¹⁴. The phosphorylated truncated tau protein can be anisoform, fragment, or a recombinant form of the protein. Likewise thephosphorylated truncated tau protein can also contain one or more aminoacid mutations. In addition to the phosphorylated truncated tau protein,the pharmaceutical composition may also contain a pharmaceutical carrierand/or a suitable adjuvant as described below. In certain embodiments,vaccination of a subject with a phosphorylated truncated tau protein, ora fragment thereof, leads to generation of antibodies that can cross theblood brain barrier and/or get produced in the brain, and subsequentlyselectively bind and react with abnormal tau and, e.g., reduce theextent of aggregated tau in the brain and slow the progression ofAlzheimer's disease or other tauopathies. In certain embodiments, thetruncated tau is phosphorylated at 1, 2, 3, 4, 5, or 6 of the followingpositions: Tyr¹⁸, Tyr²⁹, Ser¹⁸⁴, Ser¹⁸⁵, Ser¹⁹⁸, Ser¹⁹⁹, Ser²⁰², Ser²⁰⁸,Ser²¹⁴, Ser²³⁵, Ser²³⁷, Ser²³⁸, Ser²⁶², Ser²⁹³, Thr¹⁷⁵, Thr¹⁸¹, Thr²⁰⁵,Thr²¹², Thr²¹⁷, Thr²³¹, Ser⁴¹¹, Ser⁴¹², Ser⁴¹⁶, and Thr⁴¹⁴.

In certain embodiments, the truncated tau is phosphorylated at one ormore of the following amino acids: Ser¹⁹⁹, Ser²⁰², Ser²¹⁴, Ser²³⁵,Ser³⁹⁶, Ser⁴⁰⁴, Thr²⁰⁵, and Thr²¹², Ser⁴¹¹, Ser⁴¹², Ser⁴¹⁶, and Thr²¹²,Thr⁴¹⁴.

Unless otherwise indicated, reference to tau includes the natural humanamino acid sequences (SEQ ID NO: 1-6), and specifically refers to thelongest isoform of tau (SEQ ID NO: 1), also known as htau40. Variants ofsuch segments, analogs, and mimetics of the natural tau peptide thatinduce and/or crossreact with antibodies to the abnormal tau proteinscan also be used. Analogs, including allelic, species, and inducedvariants, typically differ from naturally occurring peptides at one,two, or a few positions, often by virtue of conservative substitutions.Analogs typically exhibit at least 80 or 90% sequence identity withnatural peptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids at one, two, or a fewpositions.

In addition to wildtype or natural tau proteins, the use of truncatedtau proteins containing one or more amino acid substitutions is alsocontemplated. In one embodiment of the present invention, the truncatedtau protein contains a proline to leucine mutation at amino acidposition 301 (P301L) of SEQ ID NO: 1. Other amino acid mutations of thetan protein are also contemplated. These mutations include a lysine tothreonine mutation at amino acid residue 257 (K257T) in SEQ ID NO: 1; anisoleucine to valine mutation at amino acid position 260 (1260V) of SEQID NO:1; a glycine to valine mutation at amino acid position 272 (G272V)of SEQ ID NO:1; an asparagine to lysine mutation at amino acid position279 (N279K) of SEQ ID NO:1; an asparagine to histidine mutation at aminoacid position 296 (N296H) of SEQ ID NO:1; a proline to serine mutationat amino acid position 301 (P301S) of SEQ ID NO:1; a glycine to valinemutation at amino acid position 303 (G303V) of SEQ ID NO:1; a serine toasparagine mutation at position 305 (5305N) of SEQ ID NO:1; a glycine toserine mutation at amino acid position 335 (G335S) of SEQ ID NO:1; avaline to methionine mutation at position 337 (V337M) of SEQ ID NO:1; aglutamic acid to valine mutation at position 342 (E342V) of SEQ ID NO:1;a lysine to isoleucine mutation at amino acid position 369 (K3691) ofSEQ ID NO:1; a glycine to arginine mutation at amino acid position 389(G389R) of SEQ ID NO:1; and an arginine to tryptophan mutation at aminoacid position 406 (R406W) of SEQ ID NO:1. In one embodiment of thepresent invention, the truncated tau mutant protein or peptide fragmentis phosphorylated.

Immunogenic fragments of the truncated tau protein useful for thepresent invention can be identified based on sequence antigenicity,hydrophilicity, and accessibility. In a preferred embodiment, thetruncated tau protein or its immunogenic epitopes may or may not bephosphorylated at one or more amino acids. While peptides of longerlengths have in some instances been used to successfully generateend-specific antibodies, Saido and co-workers (1993; 1994) establishedthat there is a length of five amino acids for any given peptide whichensures that the specific free amino group at the N-terminus constitutesan essential part of the epitope recognized by the new antibody. Thus,in a preferred embodiment, the immunogenic fragment of the truncated taucomprises a linear sequence of at least five amino acids of the tauprotein which ensures that the specific free amino group at theN-terminus constitutes an essential part of the epitope recognized bythe new linear epitope free-end specific antibody. In other embodiments,fragments of truncated tau have at least the last 20, 30 or 45 aminoacids of the truncated tau, and linear epitope free-end specific orconformation-specific antibodies are generated. In additionalembodiments, a fragment of a truncated tau comprises or consists of alinear sequence of four amino acids of the truncated tau.

In certain embodiments, the immunogenic fragment of the truncated taucomprises or consists of the following sequences, or fragments thereof,or a homologous sequence:

SEQ ID NO: 7 EPRQEFEVMED; SEQ ID NO: 8 PRQEFEVMED; SEQ ID NO: 9QEFEVMED; SEQ ID NO: 10 EFEVMED; SEQ ID NO: 11 FEVMED; SEQ ID NO: 12EVMED; SEQ ID NO: 13 VMED; SEQ ID NO: 14 MED; SEQ ID NO: 15HAGTYGLGDRKD; SEQ ID NO: 16 HAGTYGLGDRK; SEQ ID NO: 17 HAGTYGLGDR;SEQ ID NO: 18 HAGTYGLGD; SEQ ID NO: 19 HAGTYGLG; SEQ ID NO: 20 HAGTYGL;SEQ ID NO: 21 HAGTYG; SEQ ID NO: 22 HAGTY; SEQ ID NO: 23 HAGT;SEQ ID NO: 24 IVYKSPVVSGD; SEQ ID NO: 25 IVYKSPVVSG; SEQ ID NO: 26IVYKSPVVS; SEQ ID NO: 27 IVYKSPVV; SEQ ID NO: 28 IVYKSPV; SEQ ID NO: 29IVYKSP; SEQ ID NO: 30 IVYKS; SEQ ID NO: 31 IVYK; SEQ ID NO: 32 IVY;SEQ ID NO: 33 PQLATLADEVS SEQ ID NO: 34 PQLATLADEV; SEQ ID NO: 35PQLATLADE; SEQ ID NO: 36 PQLATLAD; SEQ ID NO: 37 PQLATLA; SEQ ID NO: 38PQLATL; SEQ ID NO: 39 PQLAT; SEQ ID NO: 40 PQLA; SEQ ID NO: 41 PQL;SEQ ID NO: 42 SPQLATLADE; SEQ ID NO: 43 SPQLATLAD; SEQ ID NO: 44SPQLATLA; SEQ ID NO: 45 SPQLATL; SEQ ID NO: 46 SPQLAT; SEQ ID NO: 47SPQLA; SEQ ID NO: 48 SPQL; SEQ ID NO: 49 SPQ; SEQ ID NO: 50 DSPQLATL;SEQ ID NO: 51 NAKAKTDHGAE; SEQ ID NO: 52 AKAKTDHGAE; SEQ ID NO: 53KAKTDHGAE; SEQ ID NO: 54 AKTDHGAE; SEQ ID NO: 55 KTDHGAE; SEQ ID NO: 56TDHGAE; SEQ ID NO: 57 DHGAE; SEQ ID NO: 58 HGAE; SEQ ID NO: 59 GAE;SEQ ID NO: 60 SSTGSIDMVDS; SEQ ID NO: 61 STGSIDMVDS; SEQ ID NO: 62TGSIDMVDS; SEQ ID NO: 63 GSIDMVDS; SEQ ID NO: 64 SIDMVDS; SEQ ID NO: 65IDMVDS; SEQ ID NO: 66 DMVDS; SEQ ID NO: 67 MVDS; SEQ ID NO: 68 VDS;SEQ ID NO: 69 NVSSTGSIDMV; SEQ ID NO: 70 VSSTGSIDMV; SEQ ID NO: 71SSTGSIDMV; SEQ ID NO: 72 STGSIDMV; SEQ ID NO: 73 TGSIDMV; SEQ ID NO: 74GSIDMV; SEQ ID NO: 75 SIDMV; SEQ ID NO: 76 IDMV; SEQ ID NO: 77 DMV;SEQ ID NO: 78 NVSTGSIDMVD; SEQ ID NO: 79 VSTGSIDMVD; SEQ ID NO: 80STGSIDMVD; SEQ ID NO: 81 TGSIDMVD; SEQ ID NO: 82 GSIDMVD; SEQ ID NO: 83SIDMVD; SEQ ID NO: 84 IDMVD; SEQ ID NO: 85 DMVD; SEQ ID NO: 86SSTGSIDMVD; SEQ ID NO: 87 SPQLATLADE; SEQ ID NO: 88 SPQLATLAD;SEQ ID NO: 89 SPQLATLA; SEQ ID NO: 90 SPQLATL; SEQ ID NO: 91 SPQLAT;SEQ ID NO: 92 SPQLA; SEQ ID NO: 93 SPQL; SEQ ID NO: 94 SPQ; andSEQ ID NO: 116.

In some of these embodiments, the immunogenic fragment of the truncatedtau comprises or consists of a sequence of SEQ ID NOS: 78-86 or 116,and, preferably, SEQ ID NO: 83-86 or 116.

The free end (N-terminus or C-terminus) of the truncated peptide is partof the immunogenic fragment and is necessary for the generation of theneoepitope-specific antibodies of the present invention (i.e., the freeN-terminus or C-terminus is an essential part of the antibody'sepitope). In certain embodiments, at least one of the serines andthreonines in these sequences is phosporylated, and the phosphorylatedserine(s) and/or threonine(s) is also an essential part of theantibody's epitope.

In certain embodiments, truncated tau comprises or consists ofphosphorylated or non-phosphorylated tau391-421, tau395-421 (e.g.,phosphorylated or non-phosphorylated at one or more of the followingSer396, Ser400, Ser404, Ser409, Ser412, Ser413, Tyr394, Thr205, and/orThr212), tau408-421 (e.g., phosphorylated non-phosphorylated at one ormore of the following Ser409, Ser412, and/or Ser413), tau414-421 (e.g.,phosphorylated or non-phosphorylated at one or more of the followingSer396, Ser400, Ser404, Ser409, Ser412, Ser413, Tyr394, Thr205, and/orThr212), tau415-421, tau416-421, tau417-421, tau418-421, or tau419-421,tau361-391, tau386-391, tau385-391, tau384-391.

In an embodiment of the present invention, the truncated tau peptides ofthe present invention can contain one or more D-amino acid residues. Theamino acids being in U-form would have the effect of enhancing thestability of the peptide. These D-amino acids can be in the same orderas the L-form of the peptide or assembled in a reverse order from theL-form sequence to maintain the overall topology of the native sequence(Ben-Yedidia et al., “A Retro-Inverso Peptide Analogue of InfluenzaVirus Hemagglutinin B-cell Epitope 91-108 Induces a Strong Mucosal andSystemic Immune Response and Confers Protection in Mice after IntranasalImmunization,” Mol Immunol. 39:323 (2002); Guichard, et al., “AntigenicMimicry of Natural L-peptides with Retro-Inverso-Peptidomimetics,” PNAS91:9765-9769 (1994); Benkirane, et al., “Antigenicity and Immunogenicityof Modified Synthetic Peptides Containing D-Amino Acid Residues,” J.Bio. Chem. 268(35):26279-26285 (1993), which are hereby incorporated byreference in their entirety).

Therapeutic agents can be longer polypeptides that include, for example,an active fragment (e.g., immunogenic portion) of tau peptide (e.g,ΔTau), together with other amino acids. For example, in certainembodiments the therapeutic agents include fusion proteins comprising asegment of tau linked, with or without a spacer, to a promiscuousT-helper cell epitope which promotes a B-cell response against thesegment of tau.

Another aspect of the present invention relates to a pharmaceuticalcomposition containing (one or more of) the immunogenic epitopes of thetruncated tau protein. In certain embodiments, immunogenic epitope oftruncated tau comprises tau391-421 (e.g., phosphorylated ornon-phosphorylated at one or more of the following Ser396, Ser400,Ser404, Ser409, Ser412, Ser413, Tyr394, Thr205, and/or Thr212),tau395-421 (e.g., phosphorylated or non-phosphorylated at one or more ofthe following Ser396, Ser400, Ser404, Ser409, Ser412, Ser413, Tyr394,Thr205, and/or Thr212), tau408-421 (e.g., phosphorylatednon-phosphorylated at one or more of the following Ser409, Ser412,and/or Ser413), tau361-391 (e.g., phosphorylated non-phosphorylated atSer396), tau411-421, tau416-421, tau 417-421, tau 418-421, tau 419-421,or a fragment of any of the foregoing. In certain embodiments, theimmunogenic epitope sequence comprises or consists of any one of SEQ IDNO: 7-94, or 116, or a fragment thereof.

Other portions or fragments of the tau protein which are suitable forpracticing the present invention may include recombinant forms ofabnormal tau protein (e.g., truncated tau) created by cleavage and/orphosphorylation of normal tau protein.

Abnormally truncated forms of human tau proteins—tauons—can be preparedby using any of numerous well known synthetic recombinant techniques.Briefly, most of the techniques which are used to transform cells,construct vectors, extract messenger RNA, prepare cDNA libraries, andthe like are widely practiced in the art, and most practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures widely practiced in the art. Abnormal tauproteins such as truncated tau can be synthesized by solid phase peptidesynthesis or recombinant expression, or can be obtained from naturalsources. Automatic peptide synthesizers are commercially available fromnumerous suppliers, such as Applied Biosystems (Foster City, Calif.).Recombinant expression systems can include bacteria, such as E. coli,yeast, insect cells, or mammalian cells. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989), which is herebyincorporated by reference in its entirety.

The most commonly used prokaryote system for the production ofrecombinant proteins remains E. coli, however, other microbial strainsmay also be used, such as Bacilli, for example Bacillus subtilis,various species of Pseudomonas, or other bacterial strains. In suchprokaryotic systems, plasmid vectors which contain replication sites andcontrol sequences derived from a species compatible with the host areused. Commonly used prokaryotic control sequences include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences.

A wide variety of eukaryotic hosts are also now available for productionof recombinant foreign proteins. As in bacteria, eukaryotic hosts may betransformed with expression systems which produce the desired proteindirectly, but more commonly, signal sequences are provided to effect thesecretion of the protein. Eukaryotic systems have the additionaladvantage that they are able to process introns which may occur in thegenomic sequences encoding proteins of higher organisms. Eucaryoticsystems also provide a variety of processing mechanisms which result in,for example, glycosylation, oxidation or derivatization of certain aminoacid residues, conformational control, and so forth.

Commonly used eukaryotic systems include yeast, insect cells, mammaliancells, avian cells, and cells of higher plants. The list is notexhaustive. Suitable promoters are available which are compatible andoperable for use in each of these host types as well are terminationsequences and enhancers, as e.g., the baculovirus polyhedron promoter.As above, promoters can be either constitutive or inducible. Forexample, in mammalian system, the MTII promoter can be induced by theaddition of heavy metal ions.

The particulars for the construction of expression systems suitable fordesired host are known to those in the art. For recombinant productionof the protein, the DNA encoding it is suitable ligated into theexpression system of choice, and the system is then transformed into thecompatible host cell which is then cultured and maintained underconditions wherein expression of the foreign gene takes place. Thetauons of this invention produced this way, are recovered from theculture, either by lysing the cells or from the culture medium asappropriate and known to those in the art.

Correct ligations for plasmid construction can be confirmed by firsttransforming a suitable host with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers depending on the mode ofplasmid construction, as is understood in the art.

In a variation of the present invention, an immunogenic peptide, such asa truncated tau, can be expressed/presented by a virus or bacteria aspart of an immunogenic composition. A nucleic acid encoding theimmunogenic peptide is incorporated into a genome or episome of thevirus or bacteria. Optionally, the nucleic acid is incorporated in sucha manner that the immunogenic peptide is expressed as a secreted proteinor as a fusion protein with an outer surface protein of a virus or atransmembrane protein of bacteria so that the peptide is displayed.Viruses or bacteria used in such methods should be nonpathogenic orattenuated. Suitable viruses include adenovirus, HSV, Venezuelan equineencephalitis virus and other alpha viruses, vesicular stomatitis virus,and other rhabdo viruses, vaccinia and fowl pox. Suitable bacteriainclude Salmonella and Shigella. Fusion of an immunogenic peptide toHBsAg of HBV is particularly suitable.

Immune responses against neurofibrillary tangles can also be induced byadministration of nucleic acids encoding segments of an abnormal taupeptide or a truncated tau, and fragments thereof, other peptideimmunogens, or antibodies and their component chains used for passiveimmunization. Such nucleic acids can be DNA or RNA. A nucleic acidsegment encoding an immunogen is typically linked to regulatoryelements, such as a promoter and enhancer, which allow expression of theDNA segment in the intended target cells of a patient. For expression inblood cells, as is desirable for induction of an immune response,promoter and enhancer elements from light or heavy chain immunoglobulingenes or the CMV major intermediate early promoter and enhancer aresuitable to direct expression. The linked regulatory elements and codingsequences are often cloned into a vector. For administration ofdouble-chain antibodies, the two chains can be cloned in the same orseparate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie et al., Cur. Opin. Genet. Develop. 3:102-109(1993), which is hereby incorporated by reference in its entirety);adenoviral vectors (Bett et al., J. Virol. 67:5911 (1993), which ishereby incorporated by reference in its entirety); adeno-associatedvirus vectors (Zhou et al., J. Exp. Med. 179:1867 (1994), which ishereby incorporated by reference in its entirety), viral vectors fromthe pox family including vaccinia virus and the avian pox viruses, viralvectors from the alpha virus genus, such as those derived from Sindbisand Semliki Forest Viruses (Dubensky et al., J. Virol 70:508-519 (1996),which is hereby incorporated by reference in its entirety), Venezuelanequine encephalitis virus (see U.S. Pat. No. 5,643,576 to Johnston etal., which is hereby incorporated by reference in its entirety) andrhabdoviruses, such as vesicular stomatitis virus (see WO 96/34625 toRose, which is hereby incorporated by reference in its entirety) andpapillomaviruses (Ohe, et al., Human Gene Therapy 6:325-333 (1995); WO94/12629 to Woo et al.; and Xiao & Brandsma, Nucleic Acids. Res.24:2630-2622 (1996), which are hereby incorporated by reference in theirentirety.

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. No. 5,208,036 to Eppstein et al., U.S. Pat. No.5,264,618 to Feigner et al., U.S. Pat. No. 5,279,833 to Rose, and U.S.Pat. No. 5,283,185 to Epand et al., which are hereby incorporated byreference in their entirety. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346 to Anderson etal., which is hereby incorporated by reference in its entirety). Suchvectors can further include facilitating agents (U.S. Pat. No. 5,593,970to Attardo et al., which is hereby incorporated by reference in itsentirety). DNA can also be administered using a gene gun (Xiao &Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996), which is herebyincorporated by reference in its entirety). The DNA encoding animmunogen is precipitated onto the surface of microscopic metal beads.The microprojectiles are accelerated with a shock wave or expandinghelium gas, and penetrate tissues to a depth of several cell layers. Forexample, the Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see WO 95/05853 to Carson et al.,which is hereby incorporated by reference in its entirety).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

A further aspect of the invention relates to a phosphorylated truncatedtau protein and a pharmaceutical composition containing thephosphorylated truncated tau protein. The phosphorylated truncated tauprotein can be an isoform, fragment, or a recombinant form of theprotein. Likewise the phosphorylated truncated tau protein can alsocontain one or more amino acid mutations. In addition to thephosphorylated truncated tau protein, the pharmaceutical compositionalso contains a pharmaceutical carrier and/or a suitable adjuvant asdescribed below.

Tau-peptides can also be produced by chemical synthesis of the aminoacid sequence of a tau-protein (Goedert et al., 1988, Proc. Natl. Acad.Sci. USA, 85:4051-4055), as predicted from the cloning and sequencing ofa cDNA coding for a tau-protein. This tau-protein sequence informationmay be utilized to predict the appropriate amino and carboxy terminaltau-peptides to be chemically synthesized using standard peptidesynthesis methods known in the art. These methods include a solid-phasemethod devised by R. Bruce Merrifield, (Erickson and Merrifield,“Solid-Phase Peptide Synthesis”, in The Proteins, Volume 2, H. Neurath &R. Hill (eds.) Academic Press, Inc., New York pp. 255-257; Merrifield,1986, “Solid phase synthesis”, Science, 242:341-347). In the solid-phasemethod, amino acids are added stepwise to a growing peptide chain thatis linked to an insoluble matrix, such as polystyrene beads. A majoradvantage of this method is that the desired product at each stage isbound to beads that can be rapidly filtered and washed and thus the needto purify intermediates is obviated. All of the reactions are carriedout in a single vessel, which eliminates losses due to repeatedtransfers of products. This solid phase method of chemical peptidesynthesis can readily be automated making it feasible to routinelysynthesize peptides containing about 50 residues in good yield andpurity (Stewart and Young, 1984, Solid Phase Peptide Synthesis, 2nd ed.,Pierce Chemical Co.; Tam et al., 1983, J. Am. Chem. Soc., 105:6442). Forexample, tau-peptides corresponding to amino acid residues 1 to 30 and331 to 352 could be synthesized.

The production of tau-peptides can further be achieved by recombinantDNA technology. For example, appropriate tau nucleotide coding sequencesmay be synthesized, cloned and expressed in appropriate host cells.Since the DNA sequence encoding for a tau-protein is known (Goeddert etal., 1988, Proc. Natl. Acad. Sci., USA 85:4051-4055), DNA probes may besynthesized by standard methods known in the art to screen cDNAlibraries prepared from brain tissue of Alzheimer's disease patients forthe specific tau-protein cDNA's. These DNA probes can further be used toisolate the entire family of tau-protein genes from these cDNA librariesusing methods which are well known to those skilled in the art. See, forexample, the techniques described in Maniatis et al., 1982, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,Chapter 7.

The polymerase chain reaction (PCR) technique can be utilized to amplifythe individual members of the tau family for subsequent cloning andexpression of tau-protein cDNAs (e.g., see U.S. Pat. Nos. 4,683,202;4,683,195; 4,889,818; Gyllensten et al., 1988, Proc. Nat'l Acad. Sci.USA, 85:7652-7656; Ochman et al., 1988, Genetics, 120:621-623; Trigliaet al., 1988, Nucl. Acids. Res., 16:8156; Frohman et al., 1988, Proc.Nat'l Acad. Sci. USA, 85:8998-9002; Loh et al., 1989, Science,243:217-220).

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing tau-proteins or fragmentsthereof coding sequences and appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See, for example, the techniques described in Maniatis etal., 1982, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y., Chapter 12.

A variety of host-expression vector systems may be utilized to expresstau-proteins or fragments thereof. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a coding sequence for a tau-protein or fragment thereof;yeast transformed with recombinant yeast expression vectors containing acoding sequence for a tau-protein or fragment thereof; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing a coding sequence for a tau-protein or fragmentthereof; or animal cell systems infected with recombinant virusexpression vectors (e.g., adenovirus, vaccinia virus) containing acoding sequence for a tau-protein or fragment thereof.

The expression elements of these vectors vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in mammalian cell systems, promoters such as theadenovirus late promoter or the vaccinia virus 7.5K promoter may beused. Promoters produced by recombinant DNA or synthetic techniques mayalso be used to provide for transcription of the inserted codingsequence for a tau-protein or fragment thereof.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987,Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning,Vol. II, IRL Press, Wash., D.C. Ch.3; and Bitter, 1987, HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology ofthe Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II. For complementation assays in yeast, cDNAs fortau-proteins or fragments thereof may be cloned into yeast episomalplasmids (YEp) which replicate autonomously in yeast due to the presenceof the yeast 2μ circle. The tau-protein or fragment thereof sequence maybe cloned behind either a constitutive yeast promoter such as ADH orLEU2 or an inducible promoter such as GAL (Cloning in Yeast, Chpt. 3, R.Rothstein In; DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover,1986, IRL Press, Wash., D.C.). Constructs may contain the 5′ and 3′non-translated regions of a cognate tau-protein mRNA or thosecorresponding to a yeast gene. YEp plasmids transform at high efficiencyand the plasmids are extremely stable. Alternatively, vectors may beused which promote integration of foreign DNA sequences into the yeastchromosome.

In certain embodiments, an insect system could be used to expresstau-proteins or fragments thereof. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.The tau-protein or fragment thereof coding sequence may be cloned intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of the polyhedrin gene results inproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (e.g., see Smith et al., 1983, J. Biol.,46:586; Smith, U.S. Pat. No. 4,215,051).

In cases where an adenovirus is used as an expression vector, thetau-protein or fragment thereof coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vivo or in vitro recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the tau-protein of fragment thereof in infected hosts. (e.g.,See Logan & Shenk, 1984, Proc. Natl. Acad. Sci., (USA) 81:3655-3659).Alternatively, the vaccinia 7.5K promoter may be used. (e.g., seeMackett et al., 1982, Proc. Natl. Acad. Sci., (USA) 79:7415-7419;Mackett et al., 1984, J. Virol., 49:857-864; Panicali et al., 1982,Proc. Natl. Acad. Sci., 79: 4927-4931).

Specific initiation signals may also be required for efficienttranslation of the inserted tau-protein or fragment thereof codingsequences. These signals include the ATG initiation codon and adjacentsequences. In cases where the entire tau-protein genome, including itsown initiation codon and adjacent sequences, are inserted into theappropriate expression vectors, no additional translational controlsignals may be needed. However, in cases where only a portion of thetau-protein coding sequence is inserted, exogenous translational controlsignals, including the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the tau-protein or fragment thereof coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bitter et al., 1987, Methods inEnzymol., 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression driven by certainpromoters can be elevated in the presence of certain inducers, (e.g.,zinc and cadmium ions for metallothionein promoters). Therefore,expression of the genetically engineered tau-protein or fragment thereofmay be controlled. This is important if the protein product of thecloned foreign gene is lethal to host cells. Furthermore, modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed.

The host cells which contain the tau-protein or fragment thereof codingsequence and which express the biologically active tau-protein orfragment thereof gene product may be identified by at least four generalapproaches: (a) DNA-DNA hybridization; (b) the presence or absence of“marker” gene functions; (c) assessing the level of transcription asmeasured by expression of tau-protein mRNA transcripts in host cells;and (d) detection of tau-protein gene products as measured byimmunoassays or by its biological activity. See U.S. Pat. No. 5,492,812,hereby incorporated by reference.

Once a recombinant that expresses a tau-protein or fragment thereof isidentified, the gene product should be analyzed. This can be achieved byassays based on the physical, immunological or functional properties ofthe product.

A tau-protein or fragment thereof should be immunoreactive whether itresults from the expression of the entire gene sequence, a portion ofthe gene sequence or from two or more gene sequences which are ligatedto direct the production of chimeric proteins. This reactivity may bedemonstrated by standard immunological techniques, such asradioimmunoprecipitation, radioimmune competition, or immunoblots.

1. Antibodies to Truncated Tau

Antibodies that specifically bind to and/or recognize any of the sixisoforms of truncated tau protein or hyperphosphorylated version thereofand do not recognize, bind or show reactivity to untruncated tau may betherapeutically effective in the context of the present invention, e.g.,to treat and/or prevent AD and/or another tauopathy.

The antibodies of the invention preferably specifically recognize theneoepitope created by cleavage of tau (i.e., the amino acid sequences ofthe free N-terminus or the free C-terminus of the peptide created bycleavage of tau), but do not recognize the same sequence of amino acidspresent internally in the normal tau protein. The antibodies of theinvention, preferably, do not inhibit caspase cleavage of tau at Asp421.In certain embodiments, the antibodies specifically recognize a sequenceout of SEQ ID No. 7-94 or 116, or any fragment thereof, in the truncatedtau, and do not recognize the same sequence of amino acid when presentin the normal tau protein. In certain embodiments, the antibodiesspecifically recognize a sequence out of SEQ ID Nos: 78-86 or 116, anddo not recognize the same sequence of amino acid when present in thenormal tau protein. The antibodies of the invention, in the preferredembodiments, are capable of crossing the blood-brain barrier (BBB) andare capable of clearing the peptides or truncated tau created bycleavage of tau and minimizing or preventing the neurofiblary tanglesformation. At the same time, these antibodies are not expected to affectbiological functions of the normal tau protein, because these sequencesin the normal tau protein are internal and do not contain a free N- orC-terminus which is necessary for the antibodies' recognition of theirepitope. These antibodies may therefore be used in the treatment and/orprevention of Alzheimer's disease and other tauopathies and in thepreparation of pharmaceutical compositions (e.g., vaccines) for thetreatment and prevention of these disorders.

In certain embodiments, the antibodies of the invention specificallyrecognize the neoepitope created by cleavage of tau (i.e., the aminoacid sequences of the free N-terminus or the free C-terminus of thepeptide created by cleavage of tau), but do not specifically recognizethe same sequence of amino acids present in the normal tau protein or donot bind sufficiently to clear normal tau or affect its function. Insome of these embodiments, the antibodies specifically recognize theneoepitope created by cleavage of tau at Asp421 (i.e., the freeC-terminus of ΔTau), but do not specifically recognize the same sequenceof amino acids present in the normal tau protein or do not bindsufficiently to clear normal tau or affect its function.

In certain embodiments, the antibodies recognize, bind or showreactivity with ΔTau, and do not recognize, bind or show reactivity withthe longest isoform of tau (i.e., htau40).

In certain embodiments, where the aim is to directly blockpolymerization of ΔTau, the antibody has a low off rate.

In certain embodiments, the antibody recognizes both a cleavage site(e.g., Asp421) and phosphorylated amino acid within ten, six, five orfour amino acids from the cleavage site.

In the preferred embodiments of the invention, the antibodies of theinvention (i) inhibit, reduce, clear and/or eliminate tau truncated atits C-terminus, e.g., at the glutamic acid residue Glu391 or at theaspartic acid residue Asp421, or its N-terminus (e.g., at the asparticacid residue Asp13), (ii) inhibit, reduce, clear and/or eliminateabnormal phosphorylated truncated tau (e.g., tau phosphorylated atSer396 and/or Ser404), and/or (iii) prevent the neurofiblary tanglesformation and/or increase clearance of the neurofiblary tangles, allwithout affecting the biological functions of the normal tau protein.

In certain embodiments, the antibody used in the methods of theinvention is conformational antibody MN423, TauC3, Tau12, 5A6, DC11,anti-cleaved-Tau (ASP421), clone C3 or structurally similar antibodies.In some of these embodiments, the antibody is TauC3, or a structurallyand/or functionally similar antibody.

It has been postulated that truncation of tau at Glu391 leads toAlzheimer's disease-specific conformational changes that are recognizedby the conformational antibody MN423 [Kovacech, et al. 2010; Novak, etal. 1989; Novak, et al. 1993; Csokava, et al. 2006; Skrabana, et al.2006; Skrabana, et al. 2007]. Another anti-tau antibody, DC11,recognizes abnormal tau proteins present in AD brains.

Further, it has been reported that antibody TauC3 specificallyrecognizes tau truncated at Asp421, whereas wild type tau containingthree microtubule-binding repeats (produced by alternative splicing,designated 3R), or tau proteins truncated at amino acid residues Glu391or Ala429 were not recognized by TauC3. (Gamblin, et al., supra).

Also, anti-cleaved-tau (ASP421), clone C3 is commercially available fromMillipore, and may be used in the methods of the present invention.Anti-cleaved-tau (ASP421) is selective for a synthetic peptidecorresponding to amino acids 412-421 (CSSTGSIDMVD) of human tau with aCys at the N-terminal end.

It is believed that up until the present invention however, there was noteaching or suggestion in the literature of using TauC3, DC11, MN423,and anti-cleaved-Tau (ASP421), clone C3 or related antibodies to treator prevent AD or another tauopathy.

It is further believed that TauC3, DC11, MN423, and anti-cleaved-Tau(ASP421), clone C3 or an antibody having a three-dimensionalshape/structure as TauC3, MN423, and anti-cleaved-Tau (ASP421) may beused in the methods of the present invention to treat and/or prevent ADor another tauopathy.

The phospho-independent antibody 5A6 is the first of three N-terminalantibodies to label early diffuse tangles. (Horowitz, 2004). Horowitz,et al. report that the Tau-12 epitope becomes unmasked as lesions assumea fibrillar morphology, and subsequently the extreme N-terminal epitopesof tau are lost from tangles of human AD brains, a process thatcorrelates temporally with the appearance of the C-terminal caspasetruncation-specific epitope at D421. Further, Horowitz, et al. reportthat caspase-6 cleaves tau in vitro at aspartic acid residue Asp421,causing loss of immunoreactivity with both Tau-12 and 5A6 antibodies.

It is believed that the blood-brain barrier (“BBB”) is compromised invarious neurodegenerative diseases such as AD, and immunologists areaware of the fact that anti-secreting cells can enter the brain andsecret antibodies locally. In healthy subjects, the BBB would berelatively impermeable to tau antibodies. As tau pathology begins,associated inflammatory changes and cellular stress may facilitateuptake of antibodies selective for truncated tau, and showing no bindingand/or reactivity with normal tau, into the brain and subsequently intoneurons, thereby allowing for the removal of pathological tau beforeand/or as it forms, which would in turn delay onset, treat or preventthe disease. The antibodies of the preferred embodiments of theinvention are capable of crossing BBB in a mammal suffering from or atrisk of developing a neurodegenerative disease (e.g., AD).

Truncated tau antibodies according to the invention may targetpathological truncated tau extracellularly, intracellularly, or both. Inextracellular targeting, the antibodies binding to their targets maydirectly promote their disassembly and may signal microglia to clear theantibody-protein complexes, thereby preventing or reducing potentialdirect or indirect toxic effect of extracellular tau aggregates.Intracellular tau may be cleared via antibody uptake, or by theantibody-mediated clearance of extracellular tau promoting secretion ofintracellular tau through a shift in equilibrium.

In certain embodiments, the antibody is specific and selectivelyrecognizes and reacts with Tau391-421, or a fragment thereof, (e.g.,phosphorylated or non-phosphorylated at one or more of the followingSer396, Ser400, Ser404, Ser409, Ser412, Ser413, Tyr394, Thr205, and/orThr212), Tau395-421 (e.g., phosphorylated or non-phosphorylated at oneor more of the following Ser396, Ser400, Ser404, Ser409, Ser412, Ser413,Tyr394, Thr205, and/or Thr212), Tau408-421, or a fragment thereof,(e.g., phosphorylated non-phosphorylated at one or more of the followingSer409, Ser412, and/or Ser413), or Tau361-391, or a fragment thereof,(e.g., phosphorylated non-phosphorylated at Ser396), and do notrecognize and show no reactivity to normal tau (e.g., untruncated tau)or the before mentioned peptides coupled/conjugated to a carrierprotein. In additional embodiments, the antibody is specific to earlyN-terminal cleavage sites of tau, for example, the Asp13 truncationsite.

In the preferred embodiments, the antibody recognizes a sequence out ofSEQ ID No. 7-94 or 116, or any fragment thereof, in the truncated tau,and do not recognize the same sequence of amino acid when presentinternally in the normal tau protein. In some of these embodiments, theantibodies recognize a sequence out of SEQ ID Nos. 78-86 or 116, and donot recognize the same sequence of amino acid when present in the normaltau protein (e.g., the longest isoform of tau protein (htau40)).

Thus, a preferred embodiment of the invention is directed toimmunotherapy (passive and/or active) against the free end epitopes oftruncated tau or the neoepitopes created by cleavage of tau (e.g., atAsp421). It is believed that immunotherapy against the free end epitopesof truncated tau clears soluble truncated tau from the brain andminimizes or prevents formation of the neurofibrillary tangles, pairedhelical filaments and/or pathological aggregation of tau. In certainembodiments, immunotherapy against the free end epitopes of truncatedtau blocks polymerization of tau directly. The immunotherapy may preventor delay memory loss and mental deterioration associated withtauopathies (e.g., AD), and in the preferred embodiments may improvecognitive or mental function in patient suffering from or at risk ofdeveloping a tauopathy (e.g., AD). This is not taught or suggested bythe literature to date. The literature also does not report where thesoluble truncated forms exist in the cell and if such forms andlocations are accessible to antibodies. This approach may be useful toclear soluble neurotoxic tau before it forms pathological tangles,microfibriles and/or aggregates, and does not depend on producingconformational antibodies to pre-formed tau tangles that are alreadycausing damage. It is believed that antibodies raised against linearsequences that recognize free ends of soluble tau proteins (e.g.,protein/peptides created by cleavage of tau) can directly inhibitpolymerization of tau.

Various procedures known in the art may be used for the production ofantibodies specific to the neoepitopes created by cleavage of thetau-protein (e.g., antibodies which recognize a sequence out of SEQ IDNo. 7-94 or 116, or any fragment thereof, in the truncated tau, and donot recognize the same sequence of amino acid when present in the normaltau protein). Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, humanized, single chain, Fab fragments and a Fabexpression library. For the production of antibodies, various hostanimals may be immunized by injection with a particular truncatedtau-protein, or a synthetic tau-peptide, or immunogenic portionsthereof, which may or may not be conjugated (e.g., to bovine serumalbumin), including but not limited to rabbits, mice, rats, etc. usingstandard immunization protocol (Taggert and Samloff, 1983). Followingthe completion of immunization, a fusion procedure may be performedusing, e.g., splenocytes from the hyperimmunized mice and an appropriatemyeloma cell-line SP2/0 Ag14 (ATCC CRL 1581, NS-1 (ATCC TIB18), orequivalent, by using, e.g., polyethylene glycol, and successful fusionproducts may be selected by means of HAT media and viable hybridomacolonies may then be grown out in well plates. The wells containingsuccessful fusion products may then be screened using, e.g., using ELISA(e.g., specificity and binding affinities) and antibodies selective forfree end specific truncated tau proteins or immunogenic portionsthereof, and showing no binding and/or no reactivity to a normal tau maybe isolated.

Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. In certainembodiments, the adjuvant is alum.

In one preferred embodiment, methods for generating antibodies that arefree end specific for internal cleavage sites of tau are the same as themethods described in U.S. Pat. No. 7,901,689 and corresponding EP PatentNo. 2203433 (Intellect “Recall-Vax”), except that the free N- orC-terminal end-specific epitope from a truncated tau, instead of anend-specific N- or C-terminal B-cell epitope of a naturally-occurringinternal peptide cleavage product of a precursor or mature proteindescribed in U.S. Pat. No. 7,901,689, is used. These patent filings,hereby incorporated by reference, provide methods for generatingantibodies that are free end specific for internal cleavage sites ofcertain proteins. In certain embodiments of the present invention, achimeric peptide or a mixture of chimeric peptides in which the free N-or C-terminal end-specific epitope from a truncated tau (instead of anend-specific N- or C-terminal B-cell epitope of a naturally-occurringinternal peptide cleavage product of a precursor or mature proteindescribed in U.S. Pat. No. 7,901,689) is fused with or without spaceramino acid residue(s) to a T helper cell epitope from a differentsource. The chimeric peptide or peptides are then used in an immunizingcomposition for immunizing a mammal against the free N-terminus or freeC-terminus of an internal peptide cleavage product which is aself-molecule of the immunized mammal (i.e., a truncated tau). Morespecifically as a preferred embodiment of the present invention, thechimeric peptide(s) have an N- or C-terminal end-specific truncated tauepitope, which is the first two to ten or the first two to five aminoacid residues of the N-terminus or the last two to ten or the last twoto five amino acid residues of the C-terminus of a truncated tau peptidefused to a T helper cell epitope. When such chimeric peptide(s) areadministered to a human individual as part of an immunizing composition,that individual will be immunized against the truncated tau peptide orpeptides from which the end-specific epitope is derived.

It is well-known that antibody responses produced by B cells to adefined region of a protein or peptide require that T helper cells ofthe immune system recognize another part of that antigen simultaneously.This is commonly referred to as B/T cell collaboration. According to oneaspect of the present invention, this phenomenon can be mimicked bymaking a synthetic chimeric peptide which contains both B and T cellepitopes in a contiguous linear sequence. Such chimeric peptides havebeen used very successfully to drive antibody production in mice,human/mice chimeras and primates (Sharma et al., 1993; Ifversen et al.,1995; O'Hern et al., 1997). In some of these embodiments, the epitopecontaining the first two to twenty, two to ten or two to five amino acidresidues of the free N-terminus or the last two to five, two to ten,five to thirty, ten to twenty five, or fifteen to twenty five amino acidresidues of the free C-terminus of a truncated tau (e.g., ΔTau) isfused, with or without spacer amino acid residues, to a known strong Thelper cell epitope to form a chimeric peptide. A non-limiting exampleof such a known strong T cell epitope is the well-studied tetanus toxoidpromiscuous epitope of SEQ ID NO:95. Immunization with the chimericpeptides(s) containing a truncated tau end-specific epitope fused withthe promiscuous T helper cell epitope of tetanus toxoid, as a preferredembodiment, should give rise to antibodies specific to that truncatedtau.

The desired anti-N-terminal or anti-C-terminal end-specific truncatedtau antibodies raised by the method for immunization according to thepresent invention are able to discriminate between a truncated tau(e.g., caspase-cleaved tau (e.g., ΔTau)) and the tau from which it isproteolytically derived (untruncated tau (e.g., the longest isoform oftau). These end-specific truncated tau antibodies bind specifically tothe terminus/end of a truncated tau to slow down, reduce or prevent theaccumulation, aggregation and/or polymerization of the truncated tau(either in soluble form, or conformationally different form than tau).

Single-chain antibodies as free end-specific molecules for the N- orC-terminus of truncated tau (e.g., ΔTau) can also be produced accordingto the present invention. These single chain antibodies can be singlechain composite polypeptides having free end-specific truncated taubinding capability and comprising a pair of amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain (linked V_(H)-V_(L), or single chain Fv). BothV_(H) and V_(L) may copy natural antibody sequences, or one or both ofthe chains may comprise a CDR construct of the type described in U.S.Pat. No. 5,091,513. The separate polypeptides analogous to the variableregions of the light and heavy chains are held together by a peptidelinker. Methods of production of such single chain antibodies, e.g.,single Fv (scFv), particularly where the DNA encoding the polypeptidestructures of the V_(H) and V_(L) chains are characterized or can bereadily ascertained by sequence analysis, may be accomplished inaccordance with the methods described, for example, in U.S. Pat. Nos.4,946,778, 5,091,513, 5,096,815, Biocca et al., 1993, Duan et al., 1994,Mhashilkar et al., 1995, Marasco et al., 1993, and Richardson et al.,1995. FIGS. 3A-3D (from Biocca et al., 1995) schematically show anintact antibody (FIG. 3A), a Fab fragment (FIG. 3B), a Fv fragmentconsisting of a non-covalently linked variable region complex (V, −V,(FIG. 3C) and a single chain Fv antibody (FIG. 3D).

Theo and co-workers (1993; 1994) established that there is a length offive amino acids for any given peptide which ensures that the specificfree group at the N-terminus constitutes an essential part of theepitope recognized by the new antibody. Thus, an antibody generatedagainst an immunogenic peptide may be or is evaluated for theselectivity of the antibody in its recognition of a free N- orC-terminus of a truncated tau protein. A competitive inhibition assay,using Enzyme-Linked Immunosorbant Assay (ELISA) or immunoprecipitationwith peptides corresponding to different regions of the truncated tauprotein, and the region immediately preceding caspase cleavage site inthe extracellular domain of tau protein, can determine the selectivityof the antibody.

Those of skill in the art will appreciate that a cysteine residue can beadded to the end of the above immunogenic peptides opposite from the endcorresponding to the free N-terminus or the free C-terminus of truncatedtau protein to facilitate coupling to a carrier protein. For example, acysteine residue may be added to peptides of any one of SEQ ID NOS:7-94, or 116 (e.g., SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 41, SEQ IDNO: 49, SEQ ID NO: 59, SEQ ID NO: 68, SEQ ID NO: 77, or SEQ ID NO: 94).Keyhole limpet hemocyanin (KLH), ovalbumin and bovine serum albumin(BSA) are non-limiting examples of proteins that can be used as carriersfor immunogens. The presence of an N-terminal or C-terminal cysteineresidue on the synthetic immunogen peptides provides a free sulfhydrylgroup for covalent coupling to a maleimide-activated protein. Aheterobifunctional reagent, such as an N-madeimido-6-aminocaproyl esteror a m-maleimidobeczoyl-N-hydroxysuccinimide ester (MBS), is used tocovalently couple the synthetic immunogenic peptide to the carrierprotein (see for example, Hartlow, E. et al., Antibodies A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1988). Commercial kits are also readily available for use in couplingpeptide antigens to maleimide-activated large carrier proteins.

The invention further provides a hybridoma cell producing monoclonalantibody, a polyclonal antibody or a single chain antibody that is freeend-specific for the free N-terminus or the C-terminus of a truncatedtau protein (e.g., tau1-13, tau14-441, tau14-391, tau391-414, tau1-391,tau1-421, tau14-421, tau14-410, tau391-410, tau14-412, tau391-412, tau14-383, tau14-381, or tau 14-355) or a fragment thereof anddiscriminates between a truncated tau protein and the tau proteinprecursor from which it is proteolytically derived. In certainembodiments, the hybridoma produces antibodies which are specific to theneoepitopes formed by the truncation of tau, the neoepitopes comprisinga sequence selected from SEQ ID No 7-94 or 116, or fragment thereof. Incertain embodiments, the hybridoma produces antibodies specific forΔTau. The hybridomas producing the monoclonal antibodies of the presentinvention are produced following the general procedures described byKohler and Milstein, Nature, 256, p. 495 (1975). In that procedure,hybridomas are prepared by fusing antibody-producing cells (typicallyspleen cells of mice previously immunized with an amyloid beta asantigen source) to cells from an immortal tumor cell line using somaticcell hybridization procedures.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humanized mice, humans, and others may be immunizedby injection with the relevant epitope or with any fragment oroligopeptide thereof, which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface-active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, KLH, and dinitrophenol. In certain embodiments, the adjuvantis alum.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses, e.g. to vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diptheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum as well.

The hybridomas resulting from the fusion process are allowed to grow.Thereafter, the resulting supernatants are screened using immunoassayprocedures to detect antibodies present in the supernatants capable ofbinding to the specific antigens.

In another embodiment, combinatorial antibody library technology, i.e.,antigen based selection from antibody libraries expressed on the surfaceof M13 filamentous phage, can be used for the generation of monoclonalantibodies and possesses a number of advantages relative to hybridomamethodologies (Huse, et al, 1989. Barbas, et al. 1991; Clackson, et at,1991: Burton and Barbas, 1994). The antibody of the invention may begenerated from phage antibody libraries. The general methodologiesinvolved in creating large combinatorial libraries using phage displaytechnology is described and disclosed in U.S. Pat. No. 5,223,409 issuedJun. 29, 1993.

Once monoclonal antibodies are generated, the selectivity and bindingaffinity (Kd) can be evaluated by ELISA, Biacore or other method. Forexample, in vitro bioassays can be performed on the antibodies to testfor the efficacy of the truncated tau-specific antibodies in blockingtau-induced cytotoxicity. In vitro bioassays can also be performed onthe antibodies to test for the lack of interference with function of thenormal tau. The antibodies selective for the truncated tau, and showingno binding and/or reactivity to normal tau may then be isolated, and,further evaluated in in vivo experiments, e.g., in transgenic AD models.The in vivo experiments, if conducted, will assess safety and efficacyof the isolated antibodies, using a variety of methods to measure safetyand efficacy, including, e.g., biochemical, neuropathological, imagingand cognitive tools.

Preferred antibodies may bind specifically to the aggregated form oftruncated tau without binding to the dissociated form. Alternatively, anantibody may bind specifically to the dissociated form without bindingto the aggregated form. An antibody may recognize other forms of tauthat accumulates in AD brain and related disorders. These forms differfrom the normal tau in terms of post-translational modification,glycation, proteolytic truncation, and racemization. Antibodies used intherapeutic methods usually have an intact constant region or at least asufficient portion of the constant region to interact with an Fcreceptor. Human isotype IgG1 is preferred because of it having thehighest affinity of human isotypes for the FcR1 receptor on phagocyticcells. Bispecific Fab fragments can also be used, in which one arm ofthe antibody has specificity for tau, and the other for an Fc receptor.Some antibodies bind to tau with a binding affinity greater than orequal to about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Antibodies useful in accordance with the present invention (e.g., thosecable of crossing the blood brain barrier) may be administered to ahuman patient who may be susceptible to or who is suffering from theformation of neurofibrillary tangles in order to selectively bind andtreat, reduce or eliminate the neurotoxicity caused by, e.g., the targettruncated tau proteins to which they selectively bind, if and when suchtruncated tau proteins form in vivo.

Alternatively, the antibodies may be expressed in the brain of a mammal(e.g., human patient), e.g., by administering an isolated immunogenicpeptide comprising or consisting of a sequence selected from SEQ ID Nos:7-94 or 116, or a fragment thereof, or a gene or a DNA molecule encodingfor the antibody.

The antibodies used in accordance with the invention may be monoclonalantibodies and derivatives thereof either native or recombinant,immobilized, free in solution or displayed on the surface of variousmolecules or bacteria, viruses, or other surfaces. The antibodies mayalso be humanized.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of tau. However, polyclonalsera can be specific to a particular segment of tau, such as tau379-408.Monoclonal antibodies bind to a specific epitope within the truncatedtau that can be a conformational or nonconformational epitope.

In some methods, multiple monoclonal antibodies having bindingspecificities to different epitopes are used. Such antibodies can beadministered sequentially or simultaneously.

In another embodiment, the antibodies will be produced in vivo, in thesubject in need, by administering of an antigen such as truncated taupeptide or fragments thereof. In certain embodiments, the antigencomprises or consists of a sequence selected from SEQ ID Nos: 7-94 or116, or a fragment thereof. The titer of the antibodies will bedetermined by techniques which are known to one skilled in the art andadditional antigen will be administered if required.

In another embodiment there is provided a pharmaceutical compositioncomprised of the antibodies described above and a method of using thecomposition for the inhibition of the formation of neurofibrillarytangles. The diminished presence of neurofibrillary tangles will delaythe progression of the Alzheimer's disease or other diseasescharacterized by tau aggregation/formation of neurofibrillary tangles,in a subject in need.

In one embodiment, the composition includes an antibody in atherapeutically or prophylactically effective amount sufficient toinhibit the neurotoxicity of truncated tau, and a pharmaceuticallyacceptable carrier.

In preferred embodiments, the antibodies according to the presentinvention inhibit the activity of the desired abnormal or truncated tauprotein intraneuronally and therefore can be used as intracellulardrugs. These antibodies preferably recognize the tauon-specificconformation of the target (e.g., truncated) tau protein withoutrecognizing normal human soluble tau. In other words, in preferredembodiments, the antibodies of the present invention bind to and havereactivity with the target abnormal or truncated tau protein and do notbind and show no reactivity with normal tau. The antibodies according tothe certain embodiments of the present invention may be said to be“specifically reactive” to the target abnormal tau protein if it iscapable of binding with that abnormal tau protein to thereby couple themolecule to the antibody. Specificity may be tested by any standard testavailable for detecting antibody specificity, e.g., ELISA tests,radioimmuo-assays, atomic force microscopy with cantilever-bound bindingpartners, etc.

In a further aspect of the invention, the antibodies according to thepresent invention may be used for the preparation of drug or apharmaceutical composition for the treatment of tauopathies such as ADby biotechnological modification into single chain molecules equippedwith targeting sequence able to deliver them into the neuroblastomacells expressing tauons, where they bind the tauons and interfere withtheir pathological effects and increase the degradation of theabnormally truncated tau proteins.

In yet another embodiment of the invention, the antibodies of thepresent invention may be conjugated to a cytoptrotective agent or anagent which will facilitate and/or improve antibody's ability to crossthe BBB. The cytoprotective agent may be an antioxidant (e.g,melatonin); and the agent which facilitates or improves antibody'sability to cross the BBB is a hydrophobic substance which is capable ofcrossing the BBB, and is generally recognized as sage (GRAS) by theUnited States Food and Drug Administration (“FDA”). The cytoprotectiveagent or the agent which facilitates or improves antibody's ability tocross the BBB may be conjugated to the antibody directly or through alinker. The linker may be selected from the group comprising orconsisting of a hydrazine linker, a disulfite linker, a thioetherlinker, a peptide linker. In certain embodiments, the antibody isspecific for ΔTau, and the cytoptrotective agent is melatonin.

2. Immunogenic Peptide

An isolated immunogenic peptide of the present invention comprises orconsists of from about 2 to about 427 amino acids. In certainembodiments, the isolated immunogenic peptide comprises or consists oftau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau14-421, tau114-410, tau391-410, tau14-412, tau391-412, tau14-383,tau14-381, tau143-355, or a fragment of any of the foregoing.

In the preferred embodiments, the immunogenic portion of the isolatedpeptide comprises or consists of an amino acid sequence which isidentical to or homologous with the amino acid sequence of theneoepitope created by cleavage of tau, e.g., at the glutamic acidresidue Glu391, at the aspartic acid residue Asp421, or at aspartic acidresidue Asp421, or a fragment of such peptide (e.g., tau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421, tau13-421, tau14-410,tau391-410, tau14-412, tau391-412, tau 14-383, tau14-381, tau 14-355, ora fragment of any of the foregoing). The immunogenic portion of theisolated peptide generally comprises from two to ten, from two to nine,from two to eight, from two to seven, from two to six, from two to five,or from two to four amino acids in a sequence identical or homologues tothe sequence of these amino acids in a neoepitope created by cleavage oftau, e.g., at the glutamic acid residue Glu391, at the aspartic acidresidue Asp421, or at the aspartic acid residue Asp13. In the preferredembodiments, the immunogenic sequence is selected from SEQ ID Nos. 7-94or 116, or a fragment thereof. In some of these preferred embodiments,the isolated immunogenic peptide comprises or consists of tau1-421(ΔTau), or a fragment thereof, wherein the immunogenic portion of thepeptide comprises or consists of SEQ ID NOs: 78-86 or 116, or a fragmentthereof. In certain embodiments, the immunogenic portion of the isolatedpeptide comprises or consists of a sequence of the last six, five, four,three or two amino acids of ΔTau. As stated above, the free end(N-terminus or C-terminus) of the isolated immunogenic peptide is partof the immunogenic fragment and is necessary for the generation of theneoepitope-specific antibodies of the present invention; and that any ofthe Serines and/or Threonines in the above-listed sequences may or maynot be phosphorylated.

In certain embodiments, the isolated immunogenic peptide is a mimotopecomprising two peptides fused together with or without spacer residues,the first peptide mimicking the structure of the neoepitope created bycleavage of tau (i.e., the amino acid sequences bound to the free N- orC-terminus portions of a peptide created by cleavage of tau) in amammal, and the second peptide mimicking the structure of a T cellepitope derived from a different source (e.g., tetanus toxoid), whichmimotope may be used for inducing an immune response in a mammal, and,in the preferred embodiments, is for use in the treatment and/orprevention of Alzheimer's disease and other tauopathies and/or in thepreparation of a pharmaceutical composition for the treatment of thesedisorders. In the preferred embodiments, the first peptide comprises orconsists of a sequence selected from SEQ ID Nos: 7-94 or 116, or afragment thereof. In some of these embodiments, the isolated immunogenicpeptide comprises or consists of tau1-421 (ΔTau), or a fragment thereof,wherein the immunogenic portion of the peptide comprises or consists ofSEQ ID NOs: 78-86 or 116, or a fragment thereof.

In certain embodiments, the isolated immunogenic peptide is a chimericpeptide(s) comprising a 2-10 or 2-5 amino acid residues sequence fromthe B cell neoepitope created by cleavage of tau (e.g., SEQ ID Nos:78-86 or 116), the neoepitope fused to, with or without a spacer aminoacid residue(s), to a promiscuous T helper cell epitope from a differentsource than the B cell neoepitope in a contiguous linear sequence,resulting in a synthetic chimeric peptide. Chimeric peptides containingboth B and T cell epitopes in a contiguous linear sequence have beenused very successfully to drive antibody production in mice, human/micechimeras and primates (Sharma et al., 1993; Ifversen et al., 1995;O'Hern et al., 1997), herein incorporated by reference.

The isolated immunogenic peptide of the invention (e.g., a mimotope, achimeric peptides, etc.) can be derived from natural sources andisolated from a mammal, such as, for example, a human, a primate, a cat,a dog, a horse, a mouse, or a rat using standard protein purificationtechniques.

The isolated immunogenic peptide (e.g., a mimotope, a chimeric peptides,etc.) may also be synthesized chemically or produced using recombinantDNA techniques. For example, the immunogenic peptide (e.g. a truncatedtau) can be synthesized by solid phase procedures well known in the art.Suitable syntheses may be performed by utilizing “T-boc” or “F-moc”procedures. Cyclic peptides can be synthesized by solid phase methodsemploying the well-known “F-moc” procedure and polyamide resin in afully automated apparatus. Alternatively, those skilled in the art willknow the necessary laboratory procedures to perform the processmanually. Techniques and procedures for solid phase synthesis aredescribed in Solid Phase Peptide Synthesis: A Practical Approach by E.Atherton and R. C. Sheppard, published by IRL at Oxford University Press(1989) and Methods in Molecular Biology, Vol. 35: Peptide SynthesisProtocols (ed. M. W. Pennington and B. M. Dunn), chapter 7, pp. 91-171by D. Andreau et al., herein incorporated by reference.

In certain embodiments, the isolated chimeric peptides of the presentinvention can be made by synthetic chemical methods which are well knownto the ordinarily skilled artisan. For example, the chimeric peptidescan be synthesized using the automated Merrifield techniques of solidphase synthesis with either t-Boc or F-moc chemistry on PeptideSynthesizers such as an Applied Biosystems Peptide Synthesizer. Aftercomplete assembly of the desired chimeric peptide, the resin is treatedaccording to standard procedures to cleave the peptide from the resinand deblock the protecting groups on the amino acid side chains. Thefree peptide is purified by HPLC and characterized biochemically, forexample, by amino acid analysis or by sequencing. Purification andcharacterization methods for peptides are well-known to one of ordinaryskill in the art. Alternatively, the longer linear chimeric peptides canbe synthesized by well-known recombinant DNA techniques. Any standardmanual on DNA technology provides detailed protocols to produce thechimeric peptides of the invention. To construct a gene encoding achimeric peptide of the present invention, the amino acid sequence isreverse transcribed into a nucleic acid sequence, and preferably usingoptimized codon usage for the organism in which the gene will beexpressed. Next, a synthetic gene is made, typically by synthesizingoverlapping oligonucleotides which encode the peptide and any regulatoryelements, if necessary. The synthetic gene is inserted in a suitablecloning vector and recombinant clones are obtained and characterized.The chimeric peptide is then expressed under suitable conditionsappropriate for the selected expression system and host, and thechimeric peptide is purified and characterized by standard methods.

Alternatively, the amino acid sequence of the isolated immunogenicpeptide (e.g., a mimotope, a chimeric peptides, etc.) can be introducedinto an expression vector that can be expressed in a suitable expressionsystem using techniques well known in the art, followed by isolation orpurification of the expressed polypeptide of interest. A variety ofbacterial, yeast, plant, mammalian, and insect expression systems areavailable in the art and any such expression system can be used.Optionally, a polynucleotide encoding the immunogenic peptide can betranslated in a cell-free translation system.

The isolated immunogenic peptide (e.g., a mimotope, chimeric peptides,etc.) can also comprise a peptide that arise as a result of theexistence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andposttranslational events. The peptide can be expressed in systems, e.g.,cultured cells, which result in substantially the same posttranslationalmodifications present as when peptide is expressed in a native cell, orin systems that result in the alteration or omission ofposttranslational modifications, e.g., glycosylation or cleavage,present when expressed in a native cell.

The isolated immunogenic peptide of the invention, in certainembodiments, can be produced as a fusion protein that contains othernon-tau or non-tau-derived amino acid sequences, such as amino acidlinkers or signal sequences or immunogenic carriers, as well as ligandsuseful in protein purification, such as glutathione-S-transferase,histidine tag, and staphylococcal protein A. More than one immunogenicpeptide of the invention can be present in a fusion protein. Theheterologous polypeptide can be fused, for example, to the N-terminus orC-terminus of the immunogenic peptide of the invention. A polypeptide ofthe disclosure can also be produced as a fusion polypeptide comprisinghomologous amino acid sequences, i.e., other tau or tau-derivedsequences.

In certain embodiments, the isolated immunogenic peptide may be linkedto an immunogenic carrier molecule to form immunogens for vaccinationprotocols. Immunogenic carrier may comprise a material which has theproperty of independently eliciting an immunogenic response in a mammaland which can be linked (e.g. covalently coupled) to the immunogenicpeptide or a portion thereof either directly via formation of peptide orester bonds between free carboxyl, amino or hydroxyl groups in thepeptide, and corresponding groups on the immunogenic carrier material,or alternatively by bonding through a conventional bifunctional linkinggroup, or as a fusion protein.

The types of carriers which may be used in the immunogenic peptides ofthe invention will be readily known to those skilled in the art. Incertain embodiments, the carrier is selected from the group comprisingor consisting of virus-like particles (VLP); serum albumins (e.g.,bovine serum albumin (BSA)); globulins; thyroglobulins; hemoglobins;hemocyanins (particularly Keyhole Limpet Hemocyanin (KLH)); proteinsextracted from ascaris, inactivated bacterial toxins or toxoids such astetanus or diptheria toxins (TT and DT) or CRM197, the purified proteinderivative of tuberculin (PPD); or Protein D from Haemophilus influenzae(PCT Publication No. WO 91/18926) or recombinant fragments thereof (forexample, Domain 1 of Fragment C of TT, or the translocation domain of DTor Protein D ⅓rd comprising the N-terminal 100 to 110 amino acids ofHaemophilus influenzae protein D (GB 9717953. 5); polylysin;polyglutamic acid; lysine-glutamic acid copolymers; copolymerscontaining lysine or ornithine; liposome carriers, or the like. Incertain embodiments, the immunogenic carrier is KLH. In anotherembodiment, the immunogenic carrier is a virus-like particle (VLP),preferably a recombinant virus-like particle.

In certain embodiments, the carrier particle is tetanus toxoidpromiscuous epitope of SEQ ID NO:95. In other embodiments, the carrierparticle is a peptide of any one of SEQ ID NOS: 96-115.

In certain embodiments, the immunogenic peptide may be coupled toimmunogenic carriers via chemical conjugation or by expression ofgenetically engineered fusion partners. The coupling does notnecessarily need to be direct, but can occur through linker sequences.More generally, in the case where antigenic peptides are fused,conjugated or otherwise attached to an immunogenic carrier, spacer orlinker sequences are typically added at one or both ends of theantigenic peptides. Such linker sequences generally comprise sequencesrecognized by the proteasome, proteases of the endosomes or othervesicular compartment of the cell.

In one embodiment, the immunogenic peptide is expressed as a fusionprotein with the immunogenic carrier. Fusion of the peptide can beeffected by insertion into the immunogenic carrier primary sequence, orby fusion to either the N- or C-terminus of the immunogenic carrier.Hereinafter, when referring to fusion proteins of a peptide to animmunogenic carrier, the fusion to either ends of the subunit sequenceor internal insertion of the peptide within the carrier sequence areencompassed. Fusion, as referred to hereinafter, may be carried out byinsertion of the immunogenic peptide into the sequence of the carrier,by substitution of part of the sequence of the carrier with theimmunogenic peptide, or by a combination of deletion, substitution orinsertions.

One skilled in the art will easily find guidance on how to constructfusion proteins using classical molecular biology techniques. Vectorsand plasmids encoding HBcAg and HBcAg fusion proteins and useful for theexpression of a HBcAg and HBcAg fusion proteins have been described(Pumpens et al., Intervirology 44:98-114 (2001), Neyrinck, S. et al.,Nature Med. 5:1157-1163 (1999)) and can be used in the practice of thisdisclosure.

Flanking amino acid residues may be added to either end of the sequenceof the isolated immunogenic peptide to be fused to either end of thesequence of the subunit of a VLP, or for internal insertion of suchpeptidic sequence into the sequence of the subunit of a VLP. Glycine andserine residues are particularly favored amino acids to be used in theflanking sequences added to the peptide to be fused. Glycine residuesconfer additional flexibility, which may diminish the potentiallydestabilizing effect of fusing a foreign sequence into the sequence of aVLP subunit.

In certain embodiments, the isolated immunogenic peptide is chemicallycoupled to an immunogenic carrier, using techniques well known in theart. Conjugation can occur to allow free movement of peptides via singlepoint conjugation (e.g. either N-terminal or C-terminal point) or aslocked down structure where both ends of peptides are conjugated toeither an immunogenic carrier protein or to a scaffold structure suchas, e.g., a VLP. Such conjugation can be carried out via conjugationchemistry known to those skilled in the art such as via cysteineresidues, lysine residues or other carboxy moieties commonly known asconjugation points such as glutamic acid or aspartic acid. Thus, forexample, for direct covalent coupling it is possible to utilize acarbodiimide, glutaraldehyde or (N-[y-malcimidobutyryloxy]succinimideester, utilizing common commercially available heterobifunctionallinkers such as CDAP and SPDP (using manufacturer's instructions).Examples of conjugation of peptides, particularly cyclized peptides, toa protein carrier via acylhydrazine peptide derivatives are described inPCT Publication No. WO 03/092714. After the coupling reaction, theimmunogen can easily be isolated and purified by means of a dialysismethod, a gel filtration method, a fractionation method etc. Peptidesterminating with a cysteine residue (preferably with a linker outsidethe cyclized region) may be conveniently conjugated to a carrier proteinvia maleimide chemistry.

3. Vaccines

A vaccine in accordance with the present invention may comprise one ormore neoepitope-specific antibodies described above or the isolatedimmunogenic peptide described above, or a fragment thereof. The vaccineis used for inducing an immunogenic response in a mammal. In thepreferred embodiments, the immunogenic response is an immunogenicreaction to pathogenic truncated tau created by cleavage and, in certainembodiments, phosphorylation of normal tau, and/or in-vivo production ofthe neoepitope-specific antibodies described above. In certainembodiments, the response also includes an immunogenic reaction topathogenic Aβ peptide(s) (e.g., immunogenic reaction to the pathogenicAβ peptide(s) and/or generation of the antibodies which are free-endspecific for pathogenic Aβ peptide(s) and do not recognize, react orbind APP), in addition to the immunogenic reaction to pathogenictruncated tau created by cleavage and, in certain embodiments,phosphorylation of normal tau, and/or in-vivo production of theneoepitope-specific antibodies described above.

In certain embodiments, the vaccine comprises the one or moreneoepitope-specific antibodies described above. The presence ofanti-neoepitope-specific antibodies for the truncated tau in the bloodand in the extracellular space, interstitial fluid and cerebrospinalfluid of the brain, where the truncated tau is present (phosphorylatedor not-phosphorylated), in certain embodiments, promotes the formationof soluble truncated tau complexes. These soluble truncated complexesmay be cleared from the central nervous system by drainage of theextracellular space, interstitial fluid and cerebrospinal fluid into thegeneral blood circulation through, e.g., the arachnoid villi of thesuperior sagittal sinus. In this manner, the truncated tau is preventedfrom aggregation into the neurofibrillary tangles. Thus, in thepreferred embodiments of the invention, the anti-neoepitope-specificantibodies for the truncated tau: (i) inhibit, reduce, clear and/oreliminate tau truncated at its C-terminus, e.g., at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, or its N-terminus(e.g., at the aspartic acid residue Asp13), (ii) inhibit, reduce, clearand/or eliminate abnormal phosphorylated truncated tau (e.g., tauphosphorylated at Ser396 and/or Ser404), and/or (iii) prevent theneurofiblary tangles formation and/or increase clearance of theneurofiblary tangles, all without affecting the biological functions ofthe normal tau protein. In additional embodiments, theanti-neoepitope-specific antibodies may directly block polymerization ofΔTau. In the preferred embodiments, the vaccine is for use in thetreatment and/or prevention of Alzheimer's disease and/or anothertauopathy.

In other embodiments, the vaccine comprises the isolated immunogenicpeptide described above, or a fragment thereof (a peptide of SEQ ID NO:7-94 or 116). The immunogenic response provided by the administration ofthe vaccine comprising the immunogenic peptide, or a fragment thereof,in the preferred embodiments, is production of the neoepitope-specificantibodies described above.

The antibodies administered or produced in the body in response to theadministration of the isolated immunogenic peptide recognize theneoepitope created by cleavage of tau (i.e., the amino acid sequences ofthe free N-terminus or the free C-terminus of the peptide created bycleavage of tau), but do not recognize the same sequence of amino acidspresent in the normal tau protein. In certain embodiments, theantibodies specifically recognize a sequence out of SEQ ID No. 7-94 or116, or any fragment thereof, in the truncated tau, and do not recognizethe same sequence of amino acid when present in the normal tau protein.In some of these embodiments, the antibodies recognize the neoepitopecreated by cleavage of tau at Asp421 (e.g., the neoepitope comprising orconsisting of a sequence selected from SEQ ID NOs: 78-86 or 116, whichmay or may not be phosphorylated). In the preferred embodiments of theinvention, the antibodies administered and/or produced in response tothe administration of the vaccine (i) inhibit, reduce, clear and/oreliminate tau truncated at its C-terminus, e.g., at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, or its N-terminus(e.g., at the aspartic acid residue Asp13), (ii) inhibit, reduce, clearand/or eliminate abnormal phosphorylated truncated tau (e.g., tauphosphorylated at Ser396 and/or Ser404), and/or (iii) prevent theneurofiblary tangles formation and/or increase clearance of theneurofiblary tangles, all without affecting the biological functions ofthe normal tau protein. In the preferred embodiments, the vaccine is foruse in the treatment and/or prevention of Alzheimer's disease and othertauopathies.

In certain embodiments, the vaccine comprises an isolated immunogenicpeptide comprising or consisting of an amino acid sequence which isidentical to or homologous with the amino acid sequence of theneoepitope created by cleavage of tau, e.g., at the glutamic acidresidue Glu391, at the aspartic acid residue Asp421, or at the asparticacid residue Asp13, or a fragment of such peptide (e.g., tau1-13, tau14-441, tau14-391, tau391-414, tau1-391, tau1-421, tau13-410,tau391-410, tau14-412, tau391-412, tau 13-383, tau13-381, tau 13-355, ora fragment of any of the foregoing). The immunogenic portion of thepeptide generally comprises from two to ten, from two to nine, from twoto eight, from two to seven, from two to six, from two to five, or fromtwo to four amino acids in a sequence identical to or homologues withthe sequence of these amino acids in a neoepitope created by cleavage oftau, e.g., at the glutamic acid residue Glu391, at the aspartic acidresidue Asp421, or at the aspartic acid residue Asp13. In the preferredembodiments, the immunogenic sequence is selected from SEQ ID Nos. 7-94or 116, or a fragment thereof. In some of these embodiments, theisolated immunogenic peptide comprises or consists of tau1-421 (ΔTau),or a fragment thereof, and the immunogenic portion of the peptidecomprises or consists of SEQ ID NOs: 78-86 or 116, or a fragmentthereof.

In certain embodiments, the truncated tau is selected from the groupconsisting of tau truncated at its C-terminus at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, or a tautruncated at its N-terminus, e.g., at the aspartic acid residue Asp13.The chimeric peptide, in certain embodiments, comprises or consists of asequence selected from SEQ ID Nos: 7-94 or 116, or a fragment thereof.In certain preferred embodiments, the truncated tau is selected from thegroup consisting of tau1-13, tau14-441, tau14-391, tau391-414, tau1-391,tau1-421, tau 13-421, tau14-410, tau391-410, tau14-412, tau391-412, tau14-383, tau14-381, tau14-355, or a fragment of any of the foregoing, ofany one of the six isoforms of the human tau protein. The truncated tau,in certain embodiments, may be phosphorylated at one or more of thefollowing: Ser199, Ser202, Ser214, Ser235, Ser396, Ser404, Thr205,Thr231, and Thr212, if present. In certain embodiments, the amino acidresidue comprises or consists of a sequence of any one of SEQ ID Nos:7-94 or 116, or a fragment thereof.

In certain preferred embodiments, the truncated tau portion of thechimeric peptide is derived from B cells. It is well-known that antibodyresponses produced by B cells to a defined region of a protein orpeptide require that T helper cells of the immune system recognizeanother part of that antigen simultaneously. This is commonly referredto as B/T cell collaboration. According to the present invention, thisphenomenon can be mimicked by making a synthetic chimeric peptide whichcontains both B and T cell epitopes in a contiguous linear sequence.

In certain embodiments, the vaccine comprises a composition (e.g., amimotope) comprising a chimeric peptide(s) comprising a 2-10 or 2-5amino acid residue from the B cell neoepitope created by cleavage oftau, the neoepitope fused to with or without a spacer amino acidresidue(s) to a promiscuous T helper cell epitope from a differentsource than the B cell neoepitope in a contiguous linear sequence,resulting in a synthetic chimeric peptide. Chimeric peptides containingboth B and T cell epitopes in a contiguous linear sequence have beenused very successfully to drive antibody production in mice, human/micechimeras and primates (Sharma et al., 1993; Ifversen et al., 1995;O'Hern et al., 1997), herein incorporated by reference. The promiscuousT helper cell epitope (T_(h)) is generally derived from a natural sourcedifferent from the source of the B neoepitope. In other words, the T_(h)epitope is not recognized as part of a self-molecule in the mammalsubject immunized according to the method of the present invention.Since truncated tau are self-molecules, they do not possess anyrecognizable T_(h) epitopes, and B cell epitopes of 2 to 10 or 2-5 aminoacid residues would lack any T cell epitopes altogether. Such epitopescan be provided, in certain embodiments, by specific sequences derivedfrom potent immunogens including, e.g., tetanus toxin, pertussis toxin,the measles virus F protein and the hepatitis B virus surface antigen(HBsAg). The T_(h) epitopes selected are preferably capable of elicitingT helper cell responses in large numbers of individuals expressingdiverse MHC haplotypes. These epitopes function in many differentindividuals of a heterogeneous population and are considered to bepromiscuous T_(h)epitopes. Promiscuous T_(h) epitopes provide anadvantage of eliciting potent antibody responses in most members ofgenetically diverse population groups.

In certain embodiments, compositions in accordance with the presentinvention comprise a chimeric peptide(s) comprising a 2-5 amino acidresidue from the free N or C terminus of a truncated tau (e.g., ΔTau)fused with or without a spacer amino acid residue(s) to a promiscuous Thelper cell epitope. The promiscuous T helper cell epitope (T_(h)) isgenerally derived from a source different than the source of thechimeric peptide. The truncated tau is selected, e.g, from the groupconsisting of tau truncated at its C-terminus at the glutamic acidresidue Glu391 or at the aspartic acid residue Asp421, or a tautruncated at its N-terminus, e.g., at the aspartic acid residue Asp13.In certain embodiments, the truncated tau is ΔTau. A non-limitingexample of such a known strong T cell epitope is the well-studiedtetanus toxoid promiscuous epitope of SEQ ID NO:95.

In certain embodiments, the vaccine is for the treatment of Alzheimer'sdisease, and comprises a mimotope fused with a bacterial peptide, themimotope mimicking the structure of the neoepitope created by cleavageof tau (i.e., the amino acid sequences bound to the free N- orC-terminus portions of a peptide created by cleavage of tau) in amammal, and the bacterial peptide comprising or consisting of a naturalbacterial tetanus toxoid or equivalent. The use of the mimotope, in thepreferred embodiments, prevents a possibility of an autoimmune responsewhich does not apply to a bacterial peptide.

In certain embodiments, the vaccine comprises a mimotope mimicking thestructure of the neoepitope created by cleavage of tau in a mammal, themimotope fused, with or without spacer residues, to a bacterial peptidecomprising or consisting of a natural bacterial tetanus toxoid orequivalent, wherein the neoepitope comprises or consists of an aminoacid sequence of amino acids 1-30, or a fragment thereof, of tau; apeptide comprising or consisting of an amino acid sequence of aminoacids 380-405, or a fragment thereof, of tau; and/or a peptidecomprising or consisting of an amino acid sequence of amino acids410-436, or a fragment thereof, of tau; and the mimitope is suitable forinducing an immunogenic response in a mammal. In some of theseembodiments, the neoepitope comprises or consists of amino acids 16-421,17-421, 18-421, or 19-421 of ΔTau. In the preferred embodiments, thevaccine is for use in a pharmaceutical composition for the treatmentand/or prevention of Alzheimer's disease and other tauopathies.

In certain preferred embodiments, the composition providing immunizationagainst truncated tau protein also comprises the composition providingimmunization against Aβ. The composition providing immunization againstAβ are, in certain embodiments, is prepared from a chimeric peptide ormixture of chimeric peptides with an end-specific B cell epitope from anaturally-occurring internal peptide cleavage product of a precursor ormature protein, as free N-terminus or C-terminus, fused with or withoutspacer amino acid residue(s) to a T helper cell epitope derived from asource different than that of the internal peptide cleavage product. Thecomposition providing immunization against Aβ are explained in detail,e.g., in the assignee's U.S. Pat. No. 7,901,689, hereby incorporated byreference in its entirety. More particularly, in such embodiments, thechimeric peptide of the present invention is represented byformula(I):N-(S)_(m)-(T_(h))_(n)  (I); orformula(II):(T_(h))_(n)-(S)_(m)-C  (II), where:N is the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues fromthe free N-terminus of a naturally-occurring internal peptide cleavageproduct of any one of the six isoforms of normal tau protein, such as,e.g., tau1-13, tau 14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau13-410, tau391-410, tau14-412, tau391-412, tau 13-383, tau13-381, tau13-355, or a fragment of any of the foregoing;C is the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from thefree C-terminus of the naturally-occurring internal peptide cleavageproduct (e.g., ΔTau) of any one of the six isoforms of normal tauprotein;T_(h) is a T helper cell epitope derived from a natural source (i.e.,species of living organism) different from that of thenaturally-occurring internal peptide cleavage product;S is a spacer amino acid residue(s);m is 0, 1, 2, 3, 4, or 5; andn is 1, 2, 3, or 4.

The embodiments, the chimeric peptide of the '689 patent is representedbyformula(I):N-(S)_(m)-(T_(h))n  (I); orformula(II):(T_(h))_(n)-(S)_(m)-C  (II), where:N is the first 2, 3, 4 or 5 amino acid residues from the free N-terminusof a naturally-occurring internal peptide cleavage product, such as anAβ peptide, which, when naturally-occurring in a mammal, is derived froma precursor protein or a mature protein;C is the last 2, 3, 4 or 5 amino acid residues from the free C-terminusof the naturally occurring internal peptide cleavage product;T_(h) is a T helper cell epitope derived from a natural source (i.e.,species of living organism) different from that of thenaturally-occurring internal peptide cleavage product;S is a spacer amino acid residue(s);m is 0, 1, 2, 3, 4, or 5; andn is 1, 2, 3, or 4.

In certain embodiments, the vaccine comprises (i) a mimotope mimickingthe structure of the neoepitope created by cleavage of tau in a mammal(e.g., at Asp421), the mimotope fused, with or without spacer residues,to a bacterial peptide comprising or consisting a structure of a T cellepitope derived from a different source (e.g., tetanus toxoid); and (ii)a mimitope mimicking the structure of the neoepitope created by cleavageof Aβ in a mammal, fused, with or without spacer residues, to abacterial peptide comprising or consisting the structure of a T cellepitope derived from a different source (e.g., tetanus toxoid). The Tcell epitope in the first mimotope and the second mimotope may be thesame or different. In certain embodiments, the T cell epitope in thefirst mimotope and in the second mimotope comprise the same structure asa well-studied tetanus toxoid promiscuous epitope of SEQ ID No: 95.

The promiscuous T helper cell epitope is, generally, a T cell epitopefrom tetanus toxin, pertussis toxin, diphtheria toxin, measles virus Fprotein, hepatitis B virus surface antigen, Chlamydia trachomitis majorouter membrane protein, Plasmodium falciparum circumsporozoite,Schistosoma mansoni triose phosphate isomerase, or Escherichia coliTraT. In certain embodiments, the promiscuous T helper cell epitope is aknown strong T cell epitope is the tetanus toxoid promiscuous epitope ofSEQ ID NO: 95.

In certain embodiments, the T helper cell epitopes in the chimericpeptide of the present invention are selected not only for a capacity tocause immune responses in most members of a given population, but alsofor a capacity to cause memory/recall responses. When the mammal ishuman, the vast majority of human subjects/patients receivingimmunotherapy with the chimeric peptide of the present invention willalready have been immunized with the pediatric vaccines (i.e., MMR anddiphtheria+pertussis+tetanus vaccines) and, possibly, the hepatitis Bvirus vaccine. These patients have therefore been previously exposed toat least one of the T_(h) epitopes present in chimeric pediatricvaccines. Prior exposure to a T_(h) epitope through immunization withthe standard vaccines should establish T_(h) cell clones which canimmediately proliferate upon administration of the chimeric peptide(i.e., a recall response), thereby stimulating rapid B cell responses tothe chimeric peptide. In addition, the T_(h) epitopes avoid anypathogen-specific B cell and/or suppressor T cell epitopes which couldlead to carrier-induced immune suppression, a problem encountered whentoxin molecules are used to elicit T helper cell responses.

The T_(h) epitopes in the chimeric peptide of the invention arepromiscuous but not universal. This characteristic means that the T_(h)epitopes are reactive in a large segment of an outbred populationexpressing different MHC antigens (reactive in 50 to 90% of thepopulation), but not in all members of that population. To provide acomprehensive, approaching universal, immune reactivity for an internalpeptide cleavage product, a combination of chimeric peptides withdifferent T_(h) epitopes can be prepared. For example, a combination offour chimeric peptides with promiscuous T_(h) epitopes from tetanus andpertussis toxins, measles virus F protein and HBsAg may be moreeffective.

Promiscuous T_(h) epitopes often share common structural features. Forexample, promiscuous T_(h) epitopes range in size from about 15 to about30 residues. Amphipathic helices are a common feature of the T_(h)epitopes. An amphipathic helix is defined by an α-helical structure withhydrophobic amino acid residues dominating the surrounding faces. T_(h)epitopes frequently contain additional primary amino acid patterns suchas a Gly or a charged reside followed by two to three hydrophobicresidues followed in turn by a charged or polar residue. This patterndefines Rothbard sequences. T_(h) epitopes often obey the 1, 4, 5, 8rule, where a positively charged residue is followed by hydrophobicresidues at the fourth, fifth and eighth positions after the chargedresidue. Since all of these structures are composed of commonhydrophobic, charged and polar amino acids, each structure can existsimultaneously within a single T_(h) epitope.

T_(h) is therefore a sequence of amino acids (natural or non-natural)that contain a T_(h) epitope. The T_(h) epitope can be a continuous ordiscontinuous epitope. Hence, not every amino acid of T_(h) isnecessarily part of the epitope. Accordingly, T_(h) epitopes, includinganalogs and segments of T_(h) epitopes, are capable of enhancing orstimulating an immune response to the internal peptide cleavage product.Immunodominant T_(h) epitopes are broadly reactive in animal and humanpopulations with widely divergent MHC types (Celis et al., 1988; Demotzet al., 1989; and Chong et al., 1992). The T_(h) domain of the chimericpeptides of the present invention has from about 10 to about 50 aminoacids residues and preferably from about 10 to about 30 amino acidsresidues. When multiple T_(h) epitopes are present, then each T_(h)epitope is independently the same or different.

T_(h) epitope analogs include substitutions, deletions and insertions ofone to about five amino acid residues in the T_(h) epitope. T_(h)segments are contiguous portions of a T_(h) epitope that are sufficientto enhance or stimulate an immune response to the internal peptidecleavage product. An example of T_(h) segments is a series ofoverlapping peptides that are derived from a single longer peptide.

The T_(h) epitopes of the present invention include, e.g., hepatitis Bsurface antigen T helper cell epitopes (HB_(s) T_(h)); pertussis toxin Thelper cell epitopes (PT T_(h)); tetanus toxin T helper cell epitopes(TT T_(h)); measles virus F protein T helper cell epitope (MV_(F1)T_(h)); Chlamydia trachomitis major outer membrane protein T helper cellepitopes (CT T_(h)); diphtheria toxin T helper cell epitopes (DT T_(h));Plasmodium falciparum circumsporozoite T helper cell epitopes (PFT_(h)); Schistosoma mansoni triose phosphate isomerase T helper cellepitopes (SM T_(h)); Escherichia coli TraT T helper cell epitopes (TraTT_(h)), and immune-enhancing analogs or any of the foregoing. Theepitopes of these T helper cells are as provided in Table 1:

TABLE 1 T helper cell Epitope TT_(O) T_(H) SEQ ID NO: 96 HB₃ T_(H) SEQID NO: 97 TT₁ T_(H) SEQ ID NO: 98 TT₁ T_(H) SEQ ID NO: 99 TT₂ T_(H) SEQID NO: 100 PT_(1A) T_(H) SEQ ID NO: 101 TT₃ T_(H) SEQ ID NO: 102 PT₂T_(H) SEQ ID NO: 103 MV_(F1) T_(H) SEQ ID NO: 104 MV_(F2) T_(H) SEQ IDNO: 105 TT₄ T_(H) SEQ ID NO: 106 TT₅ T_(H) SEQ ID NO: 107 CT₁ T_(H) SEQID NO: 108 DT₁ T_(H) SEQ ID NO: 109 DT₂ T_(H) SEQ ID NO: 110 PF T_(H)SEQ ID NO: 111 SM T_(H) SEQ ID NO: 112 TraT₁ T_(H) SEQ ID NO: 113 TraT2T_(H) SEQ ID NO: 114 TraT3 T_(H) SEQ ID NO: 115

In certain embodiments, the vaccine comprises a chimeric peptides(s)comprising a 2-10 or 2-5 amino acid residue from the B cell neoepitopecreated by cleavage of tau, the neoepitope fused to, with or without aspacer amino acid residue(s), to tetanus toxoid promiscuous epitope ofSEQ ID NO:95 in a contiguous linear sequence. Immunization with thechimeric peptides(s) comprising a 2-10 or 2-5 amino acid residue fromthe B cell neoepitope created by cleavage of tau, the neoepitope fusedto with or without a spacer amino acid residue(s) to tetanus toxoidpromiscuous epitope of SEQ ID NO:95 in a contiguous linear sequence,should give rise to the following antibodies:

(1) anti-tetanus antibody, which would be irrelevant in humans as mostindividuals are already sera-positive for tetanus toxoid (i.e., fromprevious tetanus immunizations), or which would serve as a booster forthe previous tetanus immunization;

(2) anti junction antibodies, which recognize novel epitopes created bythe junction joining the end-specific B-cell neoepitope created bycleavage of tau and the T helper cell epitope of tetanus toxoid, butwould not recognize anything other than the immunogen itself, andtherefore are not expected to produce any response in a human; and(3) anti-neoepitope-specific antibodies for the truncated tau, which arethe desired antibodies sought to be raised by the method according tothe present invention for inhibiting, reducing, or even perhapsreversing neurofibrillary tangles and/or clearing truncated tau from thebrain of a mammal. These anti-neoepitope-specific antibodies for thetruncated tau recognize the neoepitope created by cleavage of tau (i.e.,the amino acid sequences of the free N-terminus or the free C-terminusof the peptide created by cleavage of tau), but do not recognize thesame sequence of amino acids present in the normal tau protein. Incertain embodiments, the antibodies recognize a sequence out of SEQ IDNo. 7-94 or 116, or any fragment thereof, in the truncated tau, and donot recognize the same sequence of amino acid when present in the normaltau protein. In certain embodiments, these antibodies recognize (i.e.,bind and show reactivity) ΔTau, and do not recognize (i.e., bind andshow reactivity) htau40. The advantages of this method of immunizationinclude, e.g.: (1) a cheap peptide immunogen, that is readily and easilyproduced and controlled for quality assurance, is used in activeimmunization; (2) inclusion of only two to three, and perhaps up to fouror five, amino acid residues from the N- or C-terminus of an internalpeptide cleavage product of tau should minimize the amount of antibodyproduced which may react with the normal tau protein from which thetruncated tau was derived (i.e., cleaved); (3) use of an independentnon-self T cell epitope should break self-tolerance and allow productionof antibodies to a self-antigen (Schofield et al., 1991); (4) theabsence in the chimeric peptide(s) of a T cell epitope from the internalpeptide cleavage product (truncated tau) should avoid any significantproblems of autoimmunity, since anti-self T cell immunity underliesprogression of all known autoimmune diseases; and (5) the immunizationshould be self-limiting and reversible, with antibody titers graduallyfalling off with time, since the patient's immune system is not expectedto naturally encounter the combination of the truncated tau or afragment thereof with tetanus toxin as an immunogen.

In certain embodiments, the immunogenicity can be improved through theaddition of spacer residue(s) (e.g., Gly-Gly) between the promiscuousT_(h) epitope and the B cell epitope of the chimeric peptide accordingto the present invention. In addition to physically separating the T_(h)epitope from the B cell epitope, the glycine spacer residues can disruptany artificial secondary structures created by the joining of the T_(h)epitope with the B cell epitope, and thereby eliminate interferencebetween the T and/or B cell responses. The conformational separationbetween the helper epitope and the antibody eliciting domain thuspermits more efficient interactions between the presented immunogen andthe appropriate T_(h) and B cells. The amino acid residue(s) for thespacer residue(s) can be naturally-occurring amino acids ornon-naturally-occurring amino acids, which include, but are not limitedto β-alanine, ornithine, norleucine, norvaline, hydroxyproline,thyroxine, gamma-amino butyric acid, homoserine, citrulline and thelike.

In certain embodiments, an immunostimulatory epitope of the invasinprotein of a Yersinia species can be linked to the T helper cell epitopeof the chimeric peptide opposite from the B cell epitope, as an optionalsegment to the chimeric peptide. The invasions of the pathogenicbacteria Yersinia spp. are outer membrane proteins which mediate entryof the bacteria into mammalian cells (Isberg et al., 1990). Invasins ofcultured mammalian cells by the bacterium was demonstrated to requireinteraction between the Yersinia invasin molecule and several species ofthe (31 family of integrins present on the cultured cells (Tran VanNhieu et al., 1991). Since T lymphocytes are rich in (31 integrins(especially activated immune or memory T cells) the effects of invasinon human T cell have been investigated (Brett et al., 1993). It isthought that integrins facilitate the migration of immune T cells out ofthe blood vessels and through connective tissues to sites of antigenicchallenge through their interaction with extracellular matrix proteinsincluding fibronectin, laminin and collagen. The carboxy-terminus of theinvasin molecule was found to be co-stimulatory for naive human CD4⁺ Tin the presence of the non-specific mitogen, anti-CD3 antibody, causingmarked proliferation and expression of cytokines. The specific invasindomain which interacts with the β1 integrins to cause this stimulationalso was identified (Brett et al., 1993). Because of the demonstrated Tcell co-stimulatory properties associated with this domain, it can belinked to the promiscuous T_(h) epitope in the chimeric peptide of thepresent invention opposite from the B cell epitope.

In certain embodiments, a lipid common to bacterial membrane proteins,can be coupled to synthetic peptides representing either B cell orcytotoxic T cell epitopes. Many of the outer membrane proteins ofGram-negative bacteria are both lipid-modified and very immunogenic.Because of the apparent correlation between covalent lipid linkage andimmunogenicity, tripalmitoyl-S-glycerol cysteine (Pam₃Cys), a lipidcommon to bacterial membrane proteins, can be coupled to syntheticpeptides representing either B cell or cytotoxic T cell epitopes.Because significant adjuvanting responses are elicited by this lipidlinkage, promiscuous T_(h) epitope of the chimeric peptide can be lipidmodified opposite its linkage to the B cell epitope. Such lipid-modifiedchimeric peptides are likely to be more immunogenic than the unmodifiedversion of the same peptide. U.S. Pat. No. 5,843,446, which includes adisclosure of T_(h) epitopes and the immunostimulatory properties ofinvasin epitopes and lipid moieties, is herein incorporated entirely byreference.

The vaccine may optionally or preferably also include immunostimulatoryagents or adjuvants. Adjuvants have been used for many years to improvethe host immune responses, e.g. to vaccines. Intrinsic adjuvants, suchas lipopolysaccharides, normally are the components of the killed orattenuated bacteria used as vaccines. Extrinsic adjuvants areimmunomodulators which are typically non-covalently linked to antigensand are formulated to enhance the host immune responses. Thus, adjuvantshave been identified that enhance the immune response to antigensdelivered parenterally. Some of these adjuvants are toxic, however, andcan cause undesirable side-effects, making them unsuitable for use inhumans and many animals. Indeed, only aluminum hydroxide and aluminumphosphate (collectively commonly referred to as alum) are routinely usedas adjuvants in human and veterinary vaccines. The efficacy of alum inincreasing antibody responses to diptheria and tetanus toxoids is wellestablished and a HBsAg vaccine has been adjuvanted with alum as well.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes. Toefficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are emulsified in adjuvants. Many adjuvantsare toxic, inducing granulomas, acute and chronic inflammations(Freund's complete adjuvant, FCA), cytolysis (saponins and Pluronicpolymers) and pyrogenicity, arthritis and anterior uveitis (LPS andMDP). Although FCA is an excellent adjuvant and widely used in research,it is not licensed for use in human or veterinary vaccines because ofits toxicity. In certain embodiments, the adjuavant is alum.

U.S. Pat. No. 4,855,283 teaches glycolipid analogues includingN-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each ofwhich is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. U.S. Pat. No. 4,258,029 teaches thatoctadecyl tyrosine hydrochloride (OTH) functions as an adjuvant whencomplexed with tetanus toxoid and formalin inactived type I, II and IIIpoliomyelitis virus vaccine. Also, Nixon-George et al., 1990, reportedthat octadecyl esters of aromatic amino acids complexed with arecombinant hepatitis B surface antigen enhanced the host immuneresponses against hepatitis B virus.

The addition of exogenous adjuvant/emulsion formulations which maximizeimmune responses to the internal peptide cleavage product are preferred.The adjuvants and carriers that are suitable are those: (1) which havebeen successfully used in Phase I human trials; (2) based upon theirlack of reactogenicity in preclinical safety studies, have potential forapproval for use in humans; or (3) have been approved for use in foodand companion animals.

Immunotherapy regimens which produce maximal immune responses followingthe administration of the fewest number of doses, ideally only one dose,are highly desirable. This result can be approached through entrapmentof immunogen in microparticles. For example, the absorbable suturematerial poly(lactide-co-glycolide) co-polymer can be fashioned intomicroparticles containing immunogen. Following oral or parenteraladministration, microparticle hydrolysis in vivo produces the non-toxicbyproducts, lactic and glycolic acids, and releases immunogen largelyunaltered by the entrapment process. The rate of microparticledegradation and the release of entrapped immunogen can be controlled byseveral parameters, which include (1) the ratio of polymers used inparticle formation (particles with higher co-glycolide concentrationsdegrade more rapidly); (2) particle size, (smaller particles degrademore rapidly than larger ones); and, (3) entrapment efficiency,(particles with higher concentrations of entrapped antigen degrade morerapidly than particle with lower loads). Microparticle formulations canalso provide primary and subsequent booster immunizations in a singleadministration by mixing immunogen entrapped microparticles withdifferent release rates. Single dose formulations capable of releasingantigen ranging from less than one week to greater than six months canbe readily achieved. Moreover, delivery of the chimeric peptideaccording to the present invention entrapped in microparticles can alsoprovide improved efficacy when the microparticulate immunogen is mixedwith an exogenous adjuvant/emulsion formulations.

The efficacy of the chimeric peptides can be established and analyzed byinjecting an animal, e.g., mice or rats, with the chimeric peptideformulated in alum and then following the immune response to theinternal peptide cleavage product.

In certain embodiments, the vaccine contains a mixture of two or more ofthe chimeric peptides of the present invention, e.g., to enhanceimmunoefficacy in a broader population and thus provide a better immuneresponse against the truncated tau. Other immunostimulatory syntheticchimeric peptide immunogens are arrived at through modification intolipopeptides so as to provide built-in adjuvanticity for potentvaccines. The immune response to synthetic chimeric peptide immunogensof the present invention can be improved by delivery through entrapmentin or on biodegradable microparticles of the type described by O'Haganet al (1991). The immunogens can be encapsulated with or withoutadjuvant, including covalently attached lipid moiety such as Pam₃Cys,and such microparticles can be administered with an immunostimulatoryadjuvant such as Freund's Incomplete Adjuvant or alum. Themicroparticles function to potentiate immune responses to an immunogenand to provide time-controlled release for sustained or periodicresponses for oral administration, and for topical administration(O'Hagan et al., 1991).

The composition comprising an immunizing effective amount of thechimeric peptide or peptides and a pharmaceutically acceptable carrier,adjuvant, excipient, diluent, or auxiliary agent may be administered toa mammal (e.g., human) for which the truncated tau peptide is aself-molecule of the mammal.

In certain embodiments, the vaccine composition further includes achimeric peptide or mixture of chimeric peptides with an end-specific Bcell epitope from a naturally-occurring internal peptide cleavageproduct of a precursor or mature protein (e.g., APP), as free N-terminusor C-terminus, fused with or without spacer amino acid residue(s) to a Thelper cell epitope derived from a source different than that of theinternal peptide cleavage product. Such compositions are explained indetail, e.g., in the assignee's U.S. Pat. No. 7,901,689, herebyincorporated by reference in its entirety.

In certain preferred embodiments, the vaccine will comprise one or moreof the chimeric peptides of the invention and a pharmaceuticallyacceptable carrier, excipient, diluent, or auxiliary agent, includingadjuvants. The vaccine can be administered by any convenient routeincluding subcutaneous, oral, intramuscular, or other parenteral orinternal route. Similarly the vaccines can be administered as a singledose or divided into multiple doses for administration. Immunizationschedules are readily determined by the ordinary skilled artisan. Forexample, the adjuvants or emulsifiers that can be used in this inventioninclude alum, incomplete Freund's adjuvant, liposyn, saponin, squalene,L121, emulsigen and ISA720. In preferred embodiments, theadjuvants/emulsifiers are alum, incomplete Freund's adjuvant, acombination of liposyn and saponin, a combination of squalene and L121or a combination of emulsigned and saponin.

In certain embodiments, the vaccine contains a mixture of two or more ofthe neoepitope-specific antibodies described above, e.g., to enhanceimmunoefficacy in a broader population and thus provide a better immuneresponse against the truncated tau, and one or more of agent(s) selectedfrom the group comprising or consisting of pharmaceutically acceptablecarriers, adjuvants, excipients, diluents, or auxiliary agents. In someof these embodiments, the vaccine also comprises one or more antibodiesspecific the free N or C terminus of a truncated APP (e.g., Aβ₁₋₄₀,Aβ₁₋₄₂, Aβ₁₋₄₃, etc.).

4. Administration

Administration of the truncated tau protein, its immunogenic epitope, oran antibody specifically recognizing the protein or epitope, and/or avaccine described above can be used as a therapy to treat or preventAlzheimer's disease, or other tauopathy associated with the developmentof neurofibrillary tangles. Additionally, the administration of thetruncated tau protein, its immunogenic epitope and/or antibodyspecifically recognizing the protein or epitope and/or the vaccine canalso be used as a prophylactic treatment to prevent the onset ofAlzheimer's disease, or other tauopathy associated with theneurofibrillary tangle.

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease. Therefore, the presentmethods can be administered prophylactically to the general populationwithout the need for any assessment of the risk of the subject patient.Such prophylactic administration can begin at, e.g., age 50 or greater.The present methods are especially useful for individuals who do have aknown genetic risk of Alzheimer's disease. Such individuals includethose having relatives who have experienced this disease and those whoserisk is determined by analysis of genetic or biochemical markers.Genetic markers of risk toward Alzheimer's disease include mutations inthe APP gene, particularly mutations, at position 717 and positions 670and 671 referred to as the Hardy and Swedish mutations respectively.Other markers of risk are mutations in the presenilin genes, PS1 andPS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia by the presenceof risk factors described above. In addition, a number of diagnostictests are available for identifying individuals who have AD. Theseinclude imaging, and/or measurement of CSF tau and AB42 levels. Elevatedtau and decreased AB42 levels signify the presence of AD. Individualssuffering from Alzheimer's disease can also be diagnosed by Alzheimer'sDisease and Related Disorders Association criteria.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30, 40, 50, or 60). Usually, however, it is not necessary to begintreatment until a patient reaches 40, 50, 60, 70, 75 or 80. Treatmenttypically entails multiple dosages over a period of time. Treatment canbe monitored by assaying antibody, or activated T-cell or B-cellresponses to the therapeutic agent over time. If the response falls, abooster dosage is indicated. In the case of potential Down's syndromepatients, treatment can begin antenatally by administering therapeuticagent to the mother or shortly after birth.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presented duringdevelopment of the disease. In therapeutic applications, compositions ormedicaments are administered to a patient suspected of, or alreadysuffering from, such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Insome methods, administration of agent reduces or eliminates mildcognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. The amount of immunogendepends on whether adjuvant is also administered, with higher dosagesbeing required in the absence of adjuvant. An additional advantage ofthe free end specific antibodies of the present invention in certainembodiments may be that, for equal mass dosages, dosages of antibodiesthat specifically bind to neoepitopes of truncated tau (e.g., ΔTau)contain a higher molar dosage of the antibodies effective in clearingand/or “inactivating,” than a composition comprising a mixture of theneopitope-specific antibodies and non-specific antibodies. The amount ofan immunogen for administration sometimes varies from 1-500 μg perpatient and more usually from 5-500 μg per injection for humanadministration. Occasionally, a higher dose of 1-2 mg per injection isused. Typically about 10, 20, 50, or 100 μg is used for each humaninjection. The mass of immunogen also depends on the mass ratio ofimmunogenic epitope within the immunogen to the mass of immunogen as awhole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitope areused for each microgram of immunogen. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals. Another regimen consists of animmunization followed by booster injections 1, 2, and 12 months later.Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per every two weeks or once a month or onceevery 3 to 6 months. In some methods, two or more antibodies (e.g.,recombinant, monoclonal, chimeric and/or humanized) with the same ordifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. In such circumstances, the two or more antibodies mayboth be directed at, e.g., truncated tau. Alternatively, one or more ofthe antibodies may be directed at, e.g., truncated tau, and one or moreadditional antibodies may be directed at amyloid-β (AB) peptidesassociated with Alzheimer's disease. Antibodies are usually administeredon multiple occasions. Intervals between single dosages can be hourly,daily, weekly, monthly, or yearly. In some methods, dosage is adjustedto achieve a plasma antibody concentration of 1-1000 μg/ml and in somemethods 25-300 μg ml. Alternatively, antibody can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half-life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

The efficacy of the administration/treatment may be accessed bymeasuring levels of pathogenic tau in plasma and/or CSF. Based on thisassessment, the dose and/or frequency of administration may be adjustedaccordingly. In addition or in alternative, the efficacy ofadministration/treatment is accessed by monitoring the ratio of theconcentration of ΔTau to htau40, or vice versa.

In addition or in alternative, the efficacy of theadministration/treatment may also be accessed by amyloid plaques imagingby PET. An increase in brain's metabolism would indicate that theadministration/treatment is effective. The efficacy may further beaccessed by a degree of brain atrophy, as determined by MRI.

In addition or in alternative, the efficacy of theadministration/treatment may be accessed by measuring the levels of IgGand IgM against ΔTau.

The safety of the administration/treatment may be accessed by monitoringfor microhemorrhages and/vasogenic edema, e.g., by MRI. Based on thisassessment, the dose and/or frequency of administration may be adjustedaccordingly.

Antibodies and immunogens may be administered intranasally, by asubcutaneous injection, intramuscular injection, IV infusion,transcutaneously, buccally, etc., or as described in more detail below.

5. Pharmaceutical Formulations

Pharmaceutical formulations in accordance with the present invention maycomprise (i) an active agent comprising or consisting of one or moreneoepitope-specific antibody[ies] described above, one or moreimmunogenic peptide[s] described above, one or more fragment[s] of theimmunogenic peptides described above, and/or one or more mimotopesdescribed above and (ii) one or more pharmaceutically acceptableexcipients. The active agent will generally comprise from about 0.01% toabout 90% of the formulation, and the one or more excipients willgenerally comprise from about 10% to about 99.99% of the formulation. Inthe preferred embodiments, the formulations are used for introduction ofthe active agent into a body of a living mammal (e.g., a human) and areaccompanied with instructions (e.g., a package insert) which recitedirections for administration of the active agent into the body of theliving mammal. In some of these embodiments, the formulations are usedfor treatment or prevention of AD and/or another tauopathy and areaccompanied by the instructions which recited directions for treatmentand/or prevention of AD and/or another tauopathy.

In certain embodiments, the pharmaceutical formulation comprises aplurality of antibodies which recognize and bind ΔTau and do notrecognize and do not bind htau1-40, and one or more pharmaceuticallyacceptable excipients. The antibodies will generally comprise from about0.01% to about 90% of the formulation, and the one or more excipientswill generally comprise from about 10% to about 99.99% of theformulation. In the preferred embodiments, the pharmaceuticalformulation is accompanied by instructions which recite directions foradministration of the active agent into the body of the living mammaland/or directions for treatment and/or prevention of AD and/or anothertauopathy.

In certain embodiments, the pharmaceutical formulation comprises animmunogen comprising or consisting of any one of SEQ ID Nos: 7-94 or116, and one or more pharmaceutically acceptable excipients. Theimmunogen will generally comprise from about 0.01% to about 90% of theformulation, and the one or more excipients will generally comprise fromabout 10% to about 99.99% of the formulation. In the preferredembodiments, the pharmaceutical formulation is accompanied byinstructions which recite directions for administration of the activeagent into the body of the living mammal and/or directions for treatmentand/or prevention of AD and/or another tauopathy.

Formulations administered in accordance with the present invention,e.g., truncated tau proteins, portions of truncated tau proteins,immunogenic peptides, fragments of immunogenic peptides, or theneoepitope-specific antibodies described above, can be administered byparenteral, topical, intranasal, intravenous, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal, orintramuscular means for prophylactic and/or therapeutic treatment. Themost typical route of administration of an immunogenic agent issubcutaneous although other routes can be equally effective. The nextmost common route is intramuscular injection. This type of injection ismost typically performed in the arm or leg muscles. In some methods,agents are injected directly into a particular tissue where depositshave accumulated, for example intracranial injection. Intramuscularinjection on intravenous infusion are preferred for administration ofantibody. In some methods, particular therapeutic antibodies areinjected directly into the cranium. In some methods, antibodies areadministered as a sustained release composition or device, such as aMedipad™ device (Elan Pharm. Technologies, Dublin, Ireland). In certainembodiments, the adjuvant is ilum.

The pharmaceutical formulations in accordance with the present inventionmay also contain one or more pharmaceutical carriers and/or suitableadjuvants.

A therapeutically effective amount of the antibody of the invention mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the modulator to elicit adesired response in the individual. Dosage regimens may be adjusted toprovide the optimum therapeutic response. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of themodulator are outweighed by the therapeutically beneficial effects.

A “prophylactically effective amount” (e.g., of a truncated tau, aportion of truncated tau, an immunogenic peptide, a fragment of theimmunogenic peptide, or an antibody specific to any of the foregoing)refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result, such aspreventing or inhibiting the rate of tau deposition, aggregation,polymerization and/or neurotoxicity in a subject predisposed to theformation of neurofibrillary tangles. A prophylactically effectiveamount can be determined as described above for the therapeuticallyeffective amount. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic rest, such as slowed progression ofAlzheimer's disease, delayed onset, reduction or reversal of aggregateformation and/or neurofibrillary tangles, and/or reduction or reversalof neurotoxicity. A therapeutically effective amount of the antibody ofthe invention may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the modulatorto elicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the modulator are outweighed by the therapeutically beneficialeffects.

One factor that may be considered when determining a therapeutically orprophylactically effective amount of an antibody to truncated tau is theconcentration of natural tau in a biological compartment of a subject,such as in the cerebrospinal fluid (CSF) or the plasma of the subject.It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens could be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions. For example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation.

The pharmaceutical composition can be administered subcutaneously,intravenously, intradermally, intramuscularly, intraperitoneally,intracerebrally, intranasally, orally, transdermally, buccally,intra-arterially, intracranially, or intracephalically. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals. As used herein “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Preferably, the carrier canbe suitable for intravenous, intraperitoneal or intramuscularadministration. Alternatively, the carrier is suitable foradministration into the central nervous system (e.g., intraspinally orintracerebrally). In another embodiment, the carrier is suitable fororal administration. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Formulations prepared in accordance with the present invention typicallymust be sterile and stable under the conditions of manufacture andstorage. The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the antibody can be administered in atime-release formulation, for example in a composition which includes aslow release polymer. The active compounds can be prepared with carriersthat will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PLG). Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., antibody to a truncated tau in the required amount) inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Topical application can result from intransdermal or intradermalapplication. Topical administration can be facilitated bycoadministration of the agent with cholera toxin or detoxifiedderivatives or subunits thereof. Alternatively, transdermal delivery canbe achieved using skin patch or using transfersomes.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the active compounds of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acids polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. In addition, apump-based hardware delivery system can be used, some of which areadapted for implantation.

A long-term sustained release implant also may be used. “Long-term”release, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. Such implants canbe particularly useful in treating conditions characterized byaggregates of amyloid beta peptides by placing the implant near portionsof the brain affected by such aggregates, thereby effecting localized,high doses of the compounds of the invention.

Immunogenic agents of the present invention, such as peptides, aresometimes administered in combination with an adjuvant. A variety ofadjuvants can be used in combination with a peptide, such as tau, toelicit an immune response. Preferred adjuvants augment the intrinsicresponse to an immunogen without causing conformational changes in theimmunogen that affect the qualitative form of the response.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, and aluminum sulfate. Suchadjuvants can be used with or without other specific immunostimulatingagents, such as 3 De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP,polymeric or monomeric amino acids, such as polyglutamic acid orpolylysine. Such adjuvants can be used with or without other specificimmunostimulating agents, such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837 to VanNest et al., which is hereby incorporated by reference in its entirety),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (e) Ribi™ adjuvant system (RAS),(Ribi ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial c ell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Otheradjuvants include Complete Freund's Adjuvant (CFA) and IncompleteFreund's Adjuvant (IFA). Other adjuvants include cytokines, such asinterleukins (IL-1, IL-2, and IL-12), macrophage colony stimulatingfactor (M-CSF), and tumor necrosis factor (TNF). In certain embodiments,the adjuvant is ilum.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with, or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel, indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. However, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirety) alone or optionally all combinations thereof are suitable forhuman administration.

Agents of the present invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980), which is hereby incorporated by reference in its entirety. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes). Additionally, these carriers can function asimmuno stimulating agents (i.e., adjuvants).

For parenteral administration, agents of the present invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oil, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin. Peanut oil, soybean oil, and mineral oil are allexamples of useful materials. In general, glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Agents of the invention,particularly, antibodies, can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained release of the active ingredient. Anexemplary composition comprises monoclonal antibody at 5 mg/mL,formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mMNaCl, adjusted to pH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles, such as polylactide, polyglycolide, or copolymer, forenhanced adjuvant effect (Langer, et al., Science 249:1527 (1990);Hanes, et al., Advanced Drug Delivery Reviews 28:97-119 (1997), whichare hereby incorporated by reference in their entirety).

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391:851(1998), which is hereby incorporated by reference in its entirety).Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein. Alternatively, transdermal delivery can be achievedusing a skin path or using transferosomes (Paul et al., Eur. J. Immunol.25:3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368:201-15(1998), which are hereby incorporated by reference in their entirety).

6. Combination Therapy

Another aspect of the present invention is a combination therapy whereinpeptide immunogens of both truncated tau and AB (“mimotopes”) areemployed as a combination therapy to a mammal (e.g., human) in needthereof. Thus, in certain embodiments, a truncated tau protein, itsimmunogenic epitope, or antibodies specific for the truncated tauprotein or immunogenic epitope is (are) administered in combination witheach other and/or other agents that are effective for treatment ofrelated neurodegenerative diseases.

In the case of amyloidogenic diseases such as Alzheimer's disease andDown's syndrome, immune modulation to clear amyloid-beta (AB) depositsis an emerging therapy. Immunotherapies targeting AB have consistentlyresulted in cognitive improvements. It is likely that tau and ABpathologies are synergistic. Therefore, a combination therapy targetingthe clearance of both pathologies at the same time may be more effectivethan targeting each individually. In the case of Parkinson's Disease andrelated neurodegenerative diseases, immune modulation to clearaggregated forms of the α-synuclein protein is also an emerging therapy.A combination therapy which targets the clearance of both tau and-synuclein proteins simultaneously may be more effective than targetingeach individually.

In certain preferred embodiments, the therapy of the present inventionis combined with the therapies disclosed in applicant's previous filingU.S. Patent Publication No. 2003/0073655 (U.S. Ser. No. 10/084,380),hereby incorporated by reference in its entirety. That invention relatesto the use of antibodies to amyloid β peptides as a method toselectively inhibit accumulation and/or neutralize the cytotoxicityassociated with amyloid β species, and in certain preferred embodimentsspecifically AB1-40 (which forms the bulk of circulating amyloid βpeptide human CSF, plasma, and urine), or the more toxic but lessabundant AB1-42 and AB1-43 species that can seed amyloid deposition. Incertain further preferred embodiments, the invention is directed to avaccine which is a combination of a composition providing immunizationagainst truncated tau protein and a composition providing immunizationagainst Aβ.

In certain preferred embodiments, the composition providing immunizationagainst truncated tau protein and the composition providing immunizationagainst Aβ are administered to a mammal in the same or differentformulations. In certain preferred embodiments, the compositionproviding immunization against truncated tau protein and/or thecomposition providing immunization against Aβ are prepared from achimeric peptide or mixture of chimeric peptides with an end-specific Bcell epitope from a naturally-occurring internal peptide cleavageproduct of a precursor or mature protein, as free N-terminus orC-terminus, fused with or without spacer amino acid residue(s) to a Thelper cell epitope derived from a source different than that of theinternal peptide cleavage product. More particularly, in suchembodiments, the composition providing immunization against truncatedtau protein is represented byformula(I):N-(S)_(m)-(T_(h))_(n)  (I); orformula(II):(T_(h))_(n)-(S)_(m)-C  (II), where:N is the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues fromthe free N-terminus of a naturally-occurring internal peptide cleavageproduct of any one of the six isoforms of normal tau protein, such as,e.g., tau1-13, tau 14-441, tau14-391, tau391-414, tau1-391, tau1-421,tau13-410, tau391-410, tau14-412, tau391-412, tau 13-383, tau13-381, tau13-355, or a fragment of any of the foregoing;C is the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from thefree C-terminus of the naturally-occurring internal peptide cleavageproduct of any one of the six isoforms of normal tau protein;T_(h) is a T helper cell epitope derived from a natural source (i.e.,species of living organism) different from that of thenaturally-occurring internal peptide cleavage product;S is a spacer amino acid residue(s);m is 0, 1, 2, 3, 4, or 5; andn is 1, 2, 3, or 4.

The composition providing immunization against Aβ is represented byformula(I):N-(S)_(m)-(T_(h))_(n)  (I); orformula(II):(T_(h))_(n)-(S)_(m)-C  (II), where:N is the first 2, 3, 4 or 5 amino acid residues from the free N-terminusof a naturally-occurring internal peptide cleavage product, such as anAβ peptide, which, when naturally-occurring in a mammal, is derived froma precursor protein or a mature protein;C is the last 2, 3, 4 or 5 amino acid residues from the free C-terminusof the naturally occurring internal peptide cleavage product;T_(h) is a T helper cell epitope derived from a natural source (i.e.,species of living organism) different from that of thenaturally-occurring internal peptide cleavage product;S is a spacer amino acid residue(s);m is 0, 1, 2, 3, 4, or 5; andn is 1, 2, 3, or 4.

In certain embodiments, the combination therapy employs one or moreantibodies specific for the neoepitope created by cleavage of tau (e.g.,at Asp421), and one or more antibodies specific for the neoepitopecreated by cleavage of APP. The one or more antibodies specific for theneoepitope created by cleavage of tau may be any antibody describedabove in the section “Antibodies to Truncated Tau.” The one or moreantibodies specific for the neoepitope created by cleavage of APPinclude, e.g., antibodies which is specific for the free end(s) of Aβpeptides, conformation specific antibodies for these peptides andantibodies which bind to mid-domains of these peptides. In certainembodiments, the one or more antibodies specific for the neoepitopecreated by cleavage of APP is an antibody which is free end-specific forAβ peptides (Aβ1-39, Aβ1-40, Aβ1-41, Aβ1-42, Aβ1-43, etc.) and/orinternal cleavage sites at positions 11 and 17 of any of the foregoing,which may or may not have pyroglutamate modifications as a naturaloccurrence. In certain embodiments, the one or more antibodies specificfor the neoepitope created by cleavage of APP may be selected from agroup comprising or consisting of bapineuzumab, ponezumab, gantenerumab,solaneszumab, MABT5102A and GSK933756.

The efficacy of the combination treatment may be accessed by measuringAβ and pathogenic tau (e.g., ΔTau) levels in plasma and/or CSF. Inaddition or in alternative, IgG/IgM levels of Aβ and pathogenic tau(e.g., ΔTau) may be measured. In addition or in alternative, brainmetabolism may be accessed by PET imaging. In addition or inalternative, cytokine profiles from the blood may also be taken. Basedon these assessments, the dose and/or frequency of the administrationmay be adjusted.

The safety of the combination treatment may be accessed by monitoringfor microhemorrhages and/or vasogenic edema, e.g., by MRI. Based on thisassessment, the dose and/or frequency of administration may be adjustedaccordingly. For example, upon occurrence of microhemorrhages and/orvasogenic edema, the combination treatment may be temporarilydiscontinued and/or doses may be decreased.

Detailed Description of Preferred Embodiments

The following example represents specific embodiments of the presentinvention, and is not representative of the entire scope of theinvention.

Example 1

The strategy and the protocols for generating antibodies whichspecifically recognize the neoepitope created by cleavage of tau atAsp421 (i.e., ΔTau), but not full length tau (i.e., htau40) is describedin this prophetic example.

The following peptides are prepared using an Applied Biosystems PeptideSynthesizer (430A): a peptide corresponding to tau416-421 (SEQ ID NO:83SIDMVD); a peptide corresponding to tau417-421 (SEQ ID NO: 84 IDMVD); apeptide corresponding to tau 418-421 (SEQ ID NO:85 DMVD); and a peptidecorresponding to SEQ ID NO:86, all of htau40.

The synthetic peptides are then purified by HPLC and characterized usingamino acid composition.

Once purified and characterized, the peptides are conjugated to keyholelimpet hemocyanin, and four sets 10 Balb/c mice are immunized with theconjugated peptides.

Following the completion of the immunization, a fusion procedure isperformed using spleenoxytes from the hyperimmunized mice and anappropriate myeloma cell-line SP2/0-Ag14 (ATCC CRL 1581), NS-1 (ATCCTIB18), or equivalent, using polyethylene glycol.

The selection of successful fusion products are achieved by means of HATmedia. Viable hybridoma colonies are grown out in 96 well plates.

Screening of all wells containing successful fusion products are carriedout using a set of peptides corresponding to htau40, ΔTau, and residues416-421, 417-421, 418-421, and 419-421 of ΔTau by ELISA assays. Based onthe results of the ELISA assays, subclonings are performed by limitingdilution on the selected colonies. The antibodies specific for ΔTau,residues 416-421, 417-421, 418-421, and 419-421 of ΔTau; and notspecific for tau40 are selected. These antibodies only recognize, bindor show reactivity with ΔTau, or residues 416-421, 417-421, 418-421, or419-421 of ΔTau, and do not recognize, bind or show reactivity withtau40.

To confirm that the protocol is capable of being used subsequently togenerate monoclonal antibodies which specifically recognize theneoepitope created by cleavage of tau at Asp421 (i.e., ΔTau), but notfull length tau (i.e., htau40), high affinity polyclonal antibodiesspecific for ΔTau and non-specific for htau40 are raised using therestricted peptide: H2N-SEQ ID NO:86-aminohexanoate-C-amide. The peptideis synthesized using solid phase Fmoc chemistry. The peptide is thencleaved and analyzed by mass spectroscopy and high performance liquidchromatography (HPLC). HPLC purification is achieved using a C-18 YMCcolumn (10μ packing, 120 A pore size, 10×250 mm) in a buffer system ofA: H2O/0.1% TFA and B:CH3CN/0.08% TFA. The appropriate fractions ispooled, lyophilized, and again subjected to mass spectroscopy and HPLCanalysis. The peptide is coupled to KLH for immunization, BSA for ELISAdetection, with the cross-linker MBS. Rabbits are immunized at 3 weekintervals, and the titer assessed by ELISA usingacetal-Asp-Ser-aminohexanoate-C-amide (“spanning peptide”). This peptidecorresponds to a sequence of amino acid residues that spans the 0 to 1splice site of htau40 that yields ΔTau. The same spanning peptide iscoupled to a thiol coupling gel via their cysteine residue and used topreabsorb away all antibodies which do not depend upon the freecarboxy-Asp being present. The antibodies are then purified andcollected using the restricted peptide. Whereas the crude serum showssubstantial activity towards the spanning peptide, once affinitypurified, there is no reactivity of the resulting antibody with thespanning peptide, only with the restricted peptide. This confirms thatthe monoclonal antibodies are specific for ΔTau and non-specific for thespanning peptide. Since these antibodies are not specific for thespanning peptide, these antibodies would also be non-specific htau40,because htau40 also does not a free C-terminus of ΔTau, which is createdby cleavage of htau40 at Asp421. Accordingly, these antibodies onlyrecognize, bind or show reactivity with ΔTau, residues 416-421, 417-421,418-421, or 419-421 of ΔTau, and do not recognize, bind or showreactivity with tau40.

To generate monoclonal antibodies specific for the C-terminus of ΔTau,mice are immunized at 3 week intervals using: H2N-SEQ IDNO:86-aminohexanoate-C-amide conjugated to BSA prepared as described forthe preparation of polyclonal. The titer in each mouse is also assessedby ELISA as described above. After spleen cell fusion of the micecontaining the highest titer, several clones are isolated and screenedusing the spanning peptide ELISA detection method. The immunogenicpeptide sequences, corresponding to the C-terminus of tau40, andconjugated to a different carrier protein, such as bovine serum albumin(BSA) and ovalbumin, are used to confirm the resultant monoclonalantibodies are end-specific for the C-terminus of ΔTau and non-specificfor the carrier protein and htau40.

Optionally, the specificity of the antibodies to selectively recognizehtau40 cleaved at Asp421 (i.e., ΔTau), but not full length tau isassessed in vivo, using traumatic brain injury model, a model that leadsto neuronal caspase activation, in mice. Analysis shows that antibodiesspecifically detect tau cleaved at Asp421 (i.e., ΔTau), residues416-421, 417-421, 418-421, or 419-421 of tau40 and do not specificallydetect and do not cross-react with the full length tau (i.e., tau40). Inother words, these antibodies only recognize, bind or show reactivitywith ΔTau, residues 416-421, 417-421, 418-421, or 419-421 of ΔTau, anddo not recognize, bind or show reactivity with htau40.

Example 2

In Example 2, the therapeutic potential of a vaccine against Aβ, tau,and in combination in a triple transgenic mouse model of Alzheimer'sdisease (3xTg-AD) that expresses both plaque and tangle pathology isevaluated. A first goal of Example 2 is to evaluate such vaccines as apreventative against AD neuropathology and cognitive decline. The secondgoal of Example 2 is to evaluate such vaccines as a therapeutic once ADneuropathology is already established. In Example 2, the assignee'sRECALL-Vax vaccine technology is utilized.

The triple transgenic mouse model contains 3 mutations relevant toAlzheimer's pathology (PS1_(M146V), βAPP_(Swe), and tau_(P301L)) (Oddoet al., 2003). (Dr. Frank LaFerla, UC Irvine). The mice were generatedby microinjecting two transgenes (βAPPSwe, and tauP301L) into a singlecell embryo from a homozygous presenilin-1 knock-in animal. Thepresenilin-1 knock-in gene contains the mutation M146V which increasesthe amount of Aβ1-42 produced relative to Aβ1-40. Several tripletransgenic lines were derived from this approach and these lines developcritical features of Alzheimer's neuropathology in an age dependentfashion. They display plaque and tangle pathology as well as synapticdysfunction including LTP deficits (Oddo et al., 2003). Furthermoreplaque formation precedes tangle formation and so mimics the developmentof the disease in humans, and is accompanied by extensive inflammation,and substantial cognitive decline. Therefore the 3xTg-AD mice representan advanced model of AD.

Design and Methods:

Aim 1: Preventative Study on RECALL-VAX™ Vaccines in 3xTg-AD Mice.

6-month old homozygous 3xTg-AD mice will be treated with eitheradjuvant, Anti-Amyloid RECALL-VAX vaccine, Anti-ΔTau RECALL-VAX vaccineor Combination of Anti-Amyloid and Anti-ΔTau RECALL-VAX vaccine (n=20per group). RECALL-VAX™ is a proprietary vaccine owned by IntellectNeurosciences, an example of which is described and claimed in U.S. Pat.No. 7,901,689 (incorporated by reference in its entirety).

Twelve months later, these 3xTg-AD mice will be tested on a battery ofcognitive tasks, as detailed below. Afterwards, mice will be sacrificedand their brains extracted and then cut down the midline, forpathological analyses. Blood will also be taken for analyses.

Two groups of 3xTg-AD mice (n=20 per group) will be sentCEA-DSV-I2BM-MIRCen, France to utilize translational in vivo and ex vivoMRI and PET imaging approaches based on biomarkers that can bepotentially translated to clinical trials in humans. Some of thepotential biomarkers that can be used at MIRCen facility include: Brainatrophy (MRI), Amyloid plaque imaging (MRI, AV45 PET), Ab and Tau levelin plasma, Brain metabolism (FDG-PET, 2DG autoradiography), Axonaltransportation, neuronal health (MEMRI), Behavior (MWM),Microhemorrhages, Vasogenic edema (MRI).

This aim will use a total of 120 3xTg-AD mice.

Aim 2: Therapeutic Study on RECALL-VAX Vaccines in 3xTg-AD Mice.

12-month old homozygous 3xTg-AD mice will be treated with eitheradjuvant, Anti-Amyloid RECALL-VAX vaccine, Anti-ΔTau RECALL-VAX vaccineor Combination of Anti-Amyloid and Anti-ΔTau RECALL-VAX vaccine (n=15per group). Six months later, mice will be tested on a battery ofcognitive tasks, as detailed below. Afterwards, mice will be sacrificedand their brains extracted and then cut down the midline, forpathological analyses. Blood will also be taken for analyses.

This aim will use a total of 60 3xTg-AD mice.

Behavioral Assays

Object/Place/Context Recognition

These tasks are based on the spontaneous tendency of rodents to explorea novel object more often than a familiar object (Ennaceur and Delacour,1988) and have been found to not be dependent on the amygdala (Moses etal., 2005).

Perirhinal cortex lesions and studies of neuronal activation andresponses in rats suggest that it is cortical and not hippocampalneurons that are involved in the object recognition task (Aggleton etal., 1997; Wan et al., 1999). For the novel object task, mice will befamiliarized with an empty open field for a period of 10 minutes. On thefollowing day, mice will be subjected to a 5 minute exploration sessionin the same context with two identical objects (Object A; e.g. twoidentical balls or two identical dice) placed in symmetrical locationsin the open field. 90 minutes and 24 hours later, animals will besubjected to a 3 minute retention phase test where they will be exposedto one Object A and also to a novel object, Object B (for the 90 mintime point) and Object C (for the 24 hour time point) placed in thesame, symmetrical locations in the open field.

A different version of the novelty task requires mice to recognize thatan object is placed in a new location (Save et al., 1992; Ennaceur etal., 1997). This task is primarily dependent upon hippocampus (Mumby etal., 2002). For the novel place paradigm, mice will again be placed inthe open field with two identical objects (Object D), different from theobjects used for the novel object task, for 5 minutes. 90 minutes later,animals will be subjected to a 3 minute retention phase test where theywill be exposed to Objects D again but with one of the object havingbeen moved from its original location.

Another version of the novelty-preference paradigm requires mice toremember an object encountered in a particular context (Dix andAggleton, 1999). This memory task has also been shown to be hippocampusdependent (Mumby et al., 2002). The novel context task required the miceto be familiarized with a second context. Mice will be placed in asecond open field in a different room for 10 minutes. Mice will bepresented with two identical objects in context 1 (Object E) and thenpresented with two different identical objects in context 2 (Object F).For the 90 min retention phase test, animals will be placed in context 1with on Object E and one object from context 2 (Object F).

The time spent exploring the familiar object and the novel object willbe calculated where exploration equals touching the object with nose orpaws, or sniffing within 1.5 cm of the object. Time spent with the novelobject as compared to time spent with both objects will be used asmemory index. Scoring will be conducted independently by two blindscorers in order to eliminate experimental bias.

Morris Water Maze (Adapted from (Roozendaal et al., 2003))

The Morris Water Maze (MWM) is a test for spatial memory (i.e.hippocampus dependent) and cued learning (i.e. non-hippocampal) inrodents. Many studies in the last two decades have used this test as areliable measure of hippocampal-dependent learning (D'Hooge and De Deyn,2001), including several transgenic models (Hsiao et al., 1996; Hsiao,1997).

The water maze is a circular pool filled with opaque water. Mice will bepre-trained by swimming to a plexiglass platform submerged 1.5 cmbeneath the surface of the water. The location of the platform will beselected randomly each individual mouse throughout training. The maze islocated in a room containing several visual, extra-maze cues. Forspatial training, mice will be subjected to four trials per day forthree consecutive days. Before the first trial, the mouse will be placedon the platform for 30 s. On each trial, the mouse will be placed intothe tank at one of four designated staring points in a random order.Mice will be allowed to find and escape onto the submerged platform. Ifan animal fails to find the platform within 60 s, it will be manuallyguided to the platform and will remain there for 15 s.

Retention of spatial training will be assessed 1.5 and 24 hours afterthe last training trial. Both of these probe trials will consist of a 60s free swim in the pool with the platform removed. Mice will bemonitored by a camera mounted in the ceiling directly above the pool forsubsequent analysis. The parameters measured during the probe trial willinclude (1) time spent in the quadrant containing the platform duringtraining and (2) initial latency to cross platform location. The escapedata will be examined with a multifactor analysis of variance (ANOVA)including genotype (transgenic vs. non-transgenic), and probe trial (1.5and 24 hours).

Passive Inhibitory Avoidance (IA)

The inhibitory avoidance task is used in mice to assess primarilyamygdala-dependent learning (Blanchard and Blanchard, 1972; Phillips andLeDoux, 1992; Holahan and White, 2002). IA testing will consist of atraining session followed by testing 1.5 and 24 hours post trainingDuring the training session, a mouse will be placed in a lightenedchamber and when the mouse crosses to the dark compartment, it willreceive a mild footshock (0.15 mA/1 s). During testing, the mouse willbe placed again in the light compartment and the latency to cross overto the dark compartment will be measured. This latency measure will beused as an index of passive fear avoidance.

b. Biochemical Markers

AB measurements: Quantitative data on the effects of compound on variousspecies of AB (e.g. Aβ40 versus AβP42; soluble versus insoluble AB)(Oddo et al, 2003). Protein extracted from brain tissue from micetreated with compound will be used to generate soluble and insolubleprotein extracts and analyzed by sandwich ELISA. Western blots tomeasure steady state levels of the APP holoprotein, C99/C83 fragments,and sAPPβ to determine the effects of compound on these biomarkers willbe performed. Enzymatic pathways which lead to production of AB, as wellas enzymes known to degrade AB, will be looked at.

Tau hyperphosphorylation: Because the 3xTg-AD mice accumulateargyrophilic and filamentous tau immunoreactive neuronal inclusions withincreasing age in cortex and hippocampus (Oddo et al, 2003), we are ableto evaluate the effects of compound on tau hyperphosphorylation as afunctional biomarker. This will be accomplished with quantitativewestern blotting with antibodies (such as AT8, AT100, or PHF1) thatspecifically recognize hyperphosphorylated tau. Putative tau kinases andphosphatases will be looked at to see how treatment could be affectingtau phosphorylation.

c. Immunohistochemistry

To assess for total plaques and tangles and also microglial activation,3xTg-AD mouse brains will be paraformaldehyde-fixed and sectioned at 55μM. Using various antibodies against various forms of AB (1-40, 1-42 andoligomeric) and phosphorylated forms of tau, plaques and tangles can bevisualized for location and severity within the brain. In additionantibodies against CD45 will stain for microglial activation to see ifplaques and tangles still initiate an immune response. Changes insynaptic connections (PSD-95, synaptophysin etc.), and neuronal loss(NeuN, Fluorojade) will be also looked at.

Total animals required: 180 (with minimum 10-15 animals in each group).

Example 3

In Example 3, the safety and efficacy of an anti-A tau vaccine, ananti-Aβ vaccine and a combination anti-Δ and anti-Aβ vaccine will beaccessed. The anti-A tau vaccine will comprise an immunogenic peptide ofSEQ ID NO. 116 (H2N-VDDALINSTKIYSYFPSVGPSLIDMVD-OH) and alum. Theanti-Aβ vaccine will comprise an immunogenic peptide of SEQ ID NO. 117(H2N-DAEFGPSLVDDALINSTKIYSYFPSV-OH) and allum. The combination vaccinewill comprise a mixture of the immunogenic peptides of SEQ ID NOS. 116and 117, and alum. In these vaccines, alum will be used as adjuvant, andimmunogenic peptides will be used active agents.

Each vaccine will be administered to a group of LaFerla mice (i.e.,transgenic mice expressing 3 mutations relevant to Alzheimer's pathology(PS1_(M146V), βAPP_(Swe), and tau_(P301L))). There would also be acontrol group of mice which will not receive any vaccines containingpeptides of SEQ ID NO: 116 or SEQ ID NO 117. The “vaccine” administeredto the control group will comprise alum.

Behavioral studies will be conducted at 9 and 15 months, to accesscognitive functions.

Blood levels of Aβ1-40, Aβ1-42, ΔTau, htau40, IgG, IgM, and cytokineprofiles from the blood will be measured from 6 months to 9 months afteran initial administration and, then, at 12 month, 15 months and 18months after administration.

Imaging of amyloid plaques by AV-45-PET (Poisnel et al., AAICD, 2011)will be performed at 6 months, 12 months and 18 months, to access levelsof amyloid pathology.

Brain metabolism will be accessed by FDG-PEG (analysis with inputfunction taken from the heart of the animal) at 6 months, 12 months and18 months, to access clinical efficacy.

MRI imaging will be performed at 6 months, 12 months and 18 months, toaccess degree of inflammation, anatomy, to detect development of anymicrohaemorrhages and/or any vasogenic edema, cerebral anrophy, and/orto follow-up on evolution of individual plaques.

Axonal transportation will be accessed at 6 and 18 months by MRI, toaccess the level of Tau and Aβ pathology.

Plaque imaging will be performed at 6 and 18 months by MRI, usingGadolinium.

Three mice from each group, including a control group, will besacrificed at 3 months, 9 months, 12 months, and 15 months, to accessbiomarkers of amyloid and tau pathologies. All mice will be sacrificiesat 18 months, to access biomarkers of amyloid and tau pathology.

The generated data will be analyzed and, preferably, will confirm theefficacy and safety of the administered vaccines.

CONCLUSION

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims. The person skilled in the art knows how to employ themethods of the present invention for a variety of different purposeswhich all fall within the scope of protection of the present invention.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

The invention claimed is:
 1. A method of slowing the progression orameliorating a symptom of Alzheimer's disease in a human, comprisingadministering an antibody to a human in need of therapy for Alzheimer'sdisease in an amount effective to delay the onset, slow the progressionor ameliorate the symptom of Alzheimer's disease, wherein the antibodybinds an artificial peptide consisting of SEQ ID NOs. 78-86 with anequilibrium constant KD from 1×10⁻⁹ M to 1×10⁻¹¹ M, as measured by thesurface plasmon resonance assay utilizing peptide captured onstreptavidin chip, but does not bind hTau40, the antibody is TauC3 andis administered in a pharmaceutical formulation comprising (i) TauC3 and(ii) one or more pharmaceutically acceptable excipients.
 2. The methodof claim 1, wherein the antibody has an equilibrium constant KD withhTau40 which is from 1×10⁻⁴ M to 1×10⁻⁶ M, as measured by the surfaceplasmon resonance assay utilizing peptide captured on streptavidin chip.3. The method of claim 1, wherein the antibody shows no detectablebinding with hTau40, as measured by immunoblot.
 4. A method ofinhibiting tau polymerization in vivo, comprising administering to ahuman in need of therapy for Alzheimer's disease or other tauopathy aneffective amount of an antibody showing no reactivity and/or bindingwith a normal tau protein, and being administered in an amount effectiveto inhibit polymerization of tau in the brain of the human, wherein thenormal tau protein is hTau40, the antibody binds an artificial peptideconsisting of a sequence identical or homologous to SEQ ID Nos. 78-86,but does not recognize hTau40, provided that if the antibody binds asequence homologous to SEQ ID. NO. 84 or 85, the sequence homologous toSEQ ID. NO. 84 or 85 is not phosphorylated, the antibody is TauC3 and isadministered in a pharmaceutical formulation comprising (i) TauC3 and(ii) one or more pharmaceutically acceptable excipients.
 5. The methodof claim 4, wherein the antibody has an equilibrium constant KD withhTau40 which is from 1×10⁻⁴ M to 1×10⁻⁶ M, as measured by the surfaceplasmon resonance assay utilizing peptide captured on streptavidin chip.6. The method of claim 4, wherein the antibody has an equilibriumconstant KD with the sequence identical or homologous to SEQ ID Nos.78-86 of from 1×10⁻⁹ M to 1×10⁻¹¹ M, as measured by a surface plasmonresonance assay utilizing peptide captured on streptavidin chip.
 7. Themethod of claim 4, wherein the antibody shows no detectable binding withhTau40, as measured by immunoblot.
 8. The method of claim 4, wherein theantibody binds the sequence identical to SEQ ID Nos. 78-86 withequilibrium constant KD of from 1×10⁻⁹ M to 1×10⁻¹¹ M, as measured by asurface plasmon resonance assay utilizing peptide captured onstreptavidin chip.